The goal of this study was to identify the biosynthetic cluster for zwittermicin A, a novel, broad spectrum, aminopolyol antibiotic produced by Bacillus cereus. The nucleotide sequence of 2.7kb of DNA flanking the zwittermicin A self-resistance gene, zmaR, from B. cereus UW85 revealed three open reading frames (ORFs). Of these ORFs, two had sequence similarity to acyl-CoA dehydrogenases and polyketide synthases, respectively. Insertional inactivation demonstrated that orf2 is necessary for zwittermicin A production and that zmaR is necessary for high-level resistance to zwittermicin A but is not required for zwittermicin A production. Expression of ZmaR was temporally associated with zwittermicin A production. The results suggest that zmaR is part of a cluster of genes that is involved in zwittermicin A biosynthesis, representing the first biosynthetic pathway for an aminopolyol antibiotic.