<jats:title>ABSTRACT</jats:title> <jats:p> The macrotetrolides are a family of cyclic polyethers derived from tetramerization, in a stereospecific fashion, of the enantiomeric nonactic acid (NA) and its homologs. Isotope labeling experiments established that NA is of polyketide origin, and biochemical investigations demonstrated that 2-methyl-6,8-dihydroxynon-2 <jats:italic>E</jats:italic> -enoic acid can be converted into NA by a cell-free preparation from <jats:italic>Streptomyces lividans</jats:italic> that expresses <jats:italic>nonS</jats:italic> . These results lead to the hypothesis that macrotetrolide biosynthesis involves a pair of enantiospecific polyketide pathways. In this work, a 55-kb contiguous DNA region was cloned from <jats:italic>Streptomyces griseus</jats:italic> DSM40695, a 6.3-kb fragment of which was sequenced to reveal five open reading frames, including the previously reported <jats:italic>nonR</jats:italic> and <jats:italic>nonS</jats:italic> genes. Inactivation of <jats:italic>nonS</jats:italic> in vivo completely abolished macrotetrolide production. Complementation of the <jats:italic>nonS</jats:italic> mutant by the expression of <jats:italic>nonS</jats:italic> in <jats:italic>trans</jats:italic> fully restored its macrotetrolide production ability, with a distribution of individual macrotetrolides similar to that for the wild-type producer. In contrast, fermentation of the <jats:italic>nonS</jats:italic> mutant in the presence of exogenous (±)-NA resulted in the production of nonactin, monactin, and dinactin but not in the production of trinactin and tetranactin. These results prove the direct involvement of <jats:italic>nonS</jats:italic> in macrotetrolide biosynthesis. The difference in macrotetrolide production between in vivo complementation of the <jats:italic>nonS</jats:italic> mutant by the plasmid-borne <jats:italic>nonS</jats:italic> gene and fermentation of the <jats:italic>nonS</jats:italic> mutant in the presence of exogenously added (±)-NA suggests that NonS catalyzes the formation of (−)-NA and its homologs, supporting the existence of a pair of enantiospecific polyketide pathways for macrotetrolide biosynthesis in <jats:italic>S. griseus</jats:italic> . The latter should provide a model that can be used to study the mechanism by which polyketide synthase controls stereochemistry during polyketide biosynthesis.