1. Mass spectra of the trimethylsilyl derivative and the methyl ester of the N-trifluoroacetyl derivative of bacilysin indicated that the antibiotic had a molecular weight of 270. Several peaks in the spectrum of the methyl ester were consistent with the presence of an N-terminal alanine residue in the molecule. 2. The proton-magnetic-resonance spectrum of bacilysin confirmed that the antibiotic contained an epoxide group and the spin-spin splitting of the protons of the epoxide group indicated that the side chain of the epoxycyclohexanone ring was attached at C-4 and was alphabeta to the keto group. 3. The formation of an alphabeta-unsaturated ketone on reduction of bacilysin with chromous chloride also showed that the epoxide was alphabeta to the keto group. 4. The optical-rotatory-dispersion curve of bacilysin showed a positive Cotton effect. On the assumption that the reversed Octant rule for alphabeta-epoxyketones was applicable this revealed the absolute stereochemistry and enabled a definitive structure to be assigned to the molecule. 5. Similar measurements showed that substance AA1, isolated from culture supernatants, was the C-terminal amino acid of bacilysin. 6. Hydrolysis of substance P2 with leucine aminopeptidase and the mass spectrum of the methyl ester of its N-trifluoroacetyl derivative showed that this substance was l-analyl-l-alanine. 7. These results are discussed in relation to the biogenesis of bacilysin.