The dipeptide beta Cl-LAla-beta Cl-LAla is an antibacterial agent designed to utilize bacterial peptide transport for intracellular delivery of the alanine racemase inactivator beta Cl-LAla. The minimum inhibitory concentrations (MICs) for the peptide against Gram-negative species grown on enriched agar medium range from 1.56 to 12.5 micrograms/ml; MICs are increased to greater than 100 micrograms/ml when D-alanine is included in the medium, indicating that alanine racemase is, in fact, inhibited in sensitive species. When susceptible Gram-negative cells are grown on a minimal medium, D-alanine supplementation alone does not increase the MICs for beta Cl-LAla-beta Cl-LAla, but complete protection is afforded by supplementation with D-alanine, L-valine, L-leucine, and L-isoleucine. In liquid culture, the peptide is: bactericidal and lytic against Escherichia coli JSR-O growing in enriched medium or in minimal medium supplemented with the branched-chain amino acids; only inhibitory against these cells growing in minimal medium supplemented with D-alanine; and ineffective against these cells in minimal medium containing the branched-chain amino acids plus D-alanine. Cells exposed to beta Cl-LAla-beta Cl-LAla (with the protection of the four amino acids) have specific activities of both alanine racemase and transaminase B that are lower than those of cultures not treated with the peptide. Finally, E. coli JSR-O alanine racemase experiences time-dependent loss of activity when exposed to the dipeptide in the presence of aminopeptidases; the dipeptide alone is not an inactivator of the racemase in vitro. These results suggest the following mechanism of action for beta Cl-LAla-beta Cl-LAla: transport of the dipeptide into the cell; intracellular hydrolysis to give accumulation of beta Cl-LAla; and subsequent inactivation of targeted enzymes. Whether inactivation of the racemase or of the transaminase determines the pathophysiologic effects of the peptide depends on the composition of the growth medium.