<jats:title>ABSTRACT</jats:title> <jats:p> Although bacterial polyketides are of considerable biomedical interest, the molecular biology of polyketide biosynthesis in <jats:italic>Bacillus</jats:italic> spp., one of the richest bacterial sources of bioactive natural products, remains largely unexplored. Here we assign for the first time complete polyketide synthase (PKS) gene clusters to <jats:italic>Bacillus</jats:italic> antibiotics. Three giant modular PKS systems of the <jats:italic>trans</jats:italic> -acyltransferase type were identified in <jats:italic>Bacillus amyloliquefaciens</jats:italic> FZB 42. One of them, <jats:italic>pks1</jats:italic> , is an ortholog of the <jats:italic>pksX</jats:italic> operon with a previously unknown function in the sequenced model strain <jats:italic>Bacillus subtilis</jats:italic> 168, while the <jats:italic>pks2</jats:italic> and <jats:italic>pks3</jats:italic> clusters are novel gene clusters. Cassette mutagenesis combined with advanced mass spectrometric techniques such as matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-electrospray ionization mass spectrometry revealed that the <jats:italic>pks1</jats:italic> ( <jats:italic>bae</jats:italic> ) and <jats:italic>pks3</jats:italic> ( <jats:italic>dif</jats:italic> ) gene clusters encode the biosynthesis of the polyene antibiotics bacillaene and difficidin or oxydifficidin, respectively. In addition, <jats:italic>B. subtilis</jats:italic> OKB105 ( <jats:italic>pheA sfp</jats:italic> <jats:sup>0</jats:sup> ), a transformant of the <jats:italic>B. subtilis</jats:italic> 168 derivative JH642, was shown to produce bacillaene, demonstrating that the <jats:italic>pksX</jats:italic> gene cluster directs the synthesis of that polyketide.