Twelve mycobacillin-negative (My-) mutants of Bacillus subtilis B3 were isolated from an auxotrophically tagged mycobacillin producer organism. In whole-cell fermentations of some of these My- mutants a penta- and a nonapeptide accumulated; these peptides were also obtained in a cell-free system in which a new tripeptide was also detected. The amino acid composition, N- and C-terminal residues and amino acid sequence of these peptides agreed with those of equivalent segments of the mycobacillin molecule. The mycobacillin-synthesizing enzyme can be divided into three fractions that catalyse different steps in biosynthesis, and the defective enzyme fractions in the various mutant strains were identified by reconstitution experiments in vitro. The defects were further pin-pointed in mutant enzyme fractions by an ATP⇆P(i) exchange reaction and also by cell-free synthesis involving the use of membrane-bound enzyme. The defects so identified indicated the formation of tri-, penta- and nonapeptides as intermediates in the mycobacillin biosynthetic pathway.