A 4.20-kb SspI fragment from Bacillus thuringiensis G03 was cloned and sequenced. Sequencing analysis revealed two complete open reading frames (ORF; tzw1 and tzw2), and one incomplete ORF (tzw3) (GenBank accession no. EU293887). Tzw1 encodes a putative nonribosomal peptide synthetase with thiolation and condensation domains localized at the C-termini, whereas tzw2 and tzw3 encode acyl carrier protein and Acyl-CoA dehydrogenase, respectively. To investigate the function of tzw1 in zwittermicin A (ZA) biosynthesis, an in-frame deletion of 1,461 bp within tzw1 was constructed. The mutant abolished ZA production. Complementation of the mutant with cloned tzw1 restored ZA productivity. These results revealed that tzw1 is required for ZA biosynthesis in B. thuringiensis G03.