In the genome of Bacillus amyloliquefaciens FZB42, three operons pks1, pks2, and pks3 were identified which encode the biosynthesis of polyketides. pks1 and pks3 have been attributed to the production of bacillaene and difficidin/oxydifficidin, respectively, while the pks2 product remained hitherto unknown. Mass spectrometric analysis of the culture filtrates of the wild-type B. amyloliquefaciens FZB42 and mutants revealed pks2-specific metabolites. By combination of the mass spectrometric and UV/vis data with a database search, these compounds were attributed to four members of the macrolactin family, macrolactin A and D as well as 7-O-malonyl- and 7-O-succinyl-macrolactin. This conclusion was verified by the isolation and structure elucidation of macrolactin A using mass spectrometric and 2D-NMR studies. Macrolactin biosynthesis was investigated using feeding experiments with (13)C-acetate. (13)C-labelled macrolactin A revealed an alternating labelling of its carbon skeleton with (13)C, indicating that acetate/malonate was used as the sole precursor. The macrolactin structure is compatible with the domain organization of the pks2-operon. Similarly to pks1 and pks3, pks2 is a modular polyketide synthase system of type I which exhibits a trans-acyltransferase architecture using a discrete acyltransferase enzyme iteratively in the assembly of macrolactin. Finally, the potential for macrolactin production on a genetic and metabolic basis was found to be widely distributed among Bacillus amyloliquefaciens strains.