Sulfur-containing amino acid and peptide analogues of bestatin [((2S,3R)-3-amino-2-hydroxy-4-phenyl-butanoyl)-L-leucine] (1) have been synthesized and evaluated as inhibitors of aminopeptidase M (AP-M), leucine aminopeptidase (LAP), and aminopeptidase B (AP-B). The 2-thiolbestatin analogue (6) was found to be a potent inhibitor of all three aminopeptidases (AP-M, Ki = 4.4 microM; LAP, Ki = 0.55 microM; AP-B, Ki = 4.6 nM) but only a slightly better inhibitor of these aminopeptidases than the parent hydroxy-containing compound 1. Synthetic analogues of L-leucinethiol(4), a strong inhibitor of aminopeptidases, were prepared in which the carbon alpha to the thiol groups was substituted with methyl, methyl carboxylate, and carboxamide derivatives and found to be much weaker inhibitors of all aminopeptidases. A thioamide analogue of bestatin (49) is a modest inhibitor of AP-M (Ki = 40 microM), LAP (Ki = 0.33 microM), and AP-B (Ki = 2.4 microM). These results suggest that the sulfur atoms in 2-thiolbestatin and bestatin thioamide do not interact strongly with the active-site zinc atom of these aminopeptidases when the inhibitors are bound to the enzyme. These results are not consistent with proposed models for the inhibition of aminopeptidases by bestatin and related analogues.