<jats:title>ABSTRACT</jats:title> <jats:p> We report here the physiological and genetic characterization of an orphan histidine kinase (HK) (OhkA, <jats:italic>SCO1596</jats:italic> ) in <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Streptomyces coelicolor</jats:named-content> and its homolog (OhkAsav, <jats:italic>SAV_6741</jats:italic> ) in <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Streptomyces avermitilis</jats:named-content> . The physiological analysis showed that the <jats:italic>ohkA</jats:italic> mutant of <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">S. coelicolor</jats:named-content> exhibits impaired aerial mycelium formation and sporulation and overproduction of multiple antibiotics on mannitol-soy flour (MS) medium, especially actinorhodin (ACT) and calcium-dependent antibiotic (CDA), and disruption of <jats:italic>ohkAsav</jats:italic> in <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">S. avermitilis</jats:named-content> also led to the similar phenotypes of impaired morphological differentiation and significantly increased oligomycin A production. DNA microarray analysis combined with real-time reverse transcription-PCR (RT-PCR) and RNA dot blot assay in the <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">S. coelicolor ohkA</jats:named-content> deletion mutant confirmed the physiological results by showing the upregulation of genes involved in the biosynthesis of ACT, CDA, undecylprodigiosin (RED), a yellow type I polyketide (CPK, <jats:italic>SCO6273-6289</jats:italic> ), and a sesquiterpene antibiotic, albaflavenone ( <jats:italic>SCO5222-5223</jats:italic> ). The results also suggested that the increased production of ACT and RED in the mutant could be partly ascribed to the enhanced precursor malonyl coenzyme A (malonyl-CoA) supply through increased transcription of genes encoding acetyl-CoA carboxylase (ACCase). Interestingly, DNA microarray analysis also showed that deletion of <jats:italic>ohkA</jats:italic> greatly downregulated the transcription of <jats:italic>chpABCDEFGH</jats:italic> genes essential for aerial mycelium formation by <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">S. coelicolor</jats:named-content> on MS medium but significantly increased transcription of <jats:italic>ramS</jats:italic> / <jats:italic>C</jats:italic> / <jats:italic>R</jats:italic> , which is responsible for SapB formation and regulation and is normally absent on MS medium. Moreover, many other genes involved in development, such as <jats:italic>bldM</jats:italic> / <jats:italic>N</jats:italic> , <jats:italic>whiG</jats:italic> / <jats:italic>H</jats:italic> / <jats:italic>I</jats:italic> , <jats:italic>ssgA</jats:italic> / <jats:italic>B</jats:italic> / <jats:italic>E</jats:italic> / <jats:italic>G</jats:italic> / <jats:italic>R</jats:italic> , and <jats:italic>whiE</jats:italic> , were also significantly downregulated upon <jats:italic>ohkA</jats:italic> deletion. The results clearly demonstrated that OhkA is an important global regulator for both morphological differentiation and secondary metabolism in <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">S. coelicolor</jats:named-content> and <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">S. avermitilis</jats:named-content> .