Syntheses of several tripeptide analogues of leupeptin containing C-terminal argininal, lysinal, or ornithinal units are presented. The synthetic analogues were tested as inhibitors of trypsin, plasmin, and kallikrein. (Benzyloxycarbonyl)-L-leucyl-L-leucyl-L-argininal (2a) was significantly less effective as an inhibitor of trypsin and plasmin activity than leupeptin. (Benzyloxycarbonyl)-L-leucyl-L-leucyl-L-lysinal (2e) and (benzyloxycarbonyl)-L-leucyl-L-leucyl-L-ornithinal (2i) display different inhibition characteristics than (benzyloxycarbonyl)-L-leucyl-L-leucyl-L-argininal (2a). While (benzyloxycarbonyl)-L-leucyl-L-leucyl-L-argininal (2a) showed moderate inhibition of all three enzymes tested, (benzyloxycarbonyl)-L-leucyl-L-leucyl-L-lysinal (2e) was less effective as an inhibitor of trypsin and plasmin activity. Of the three enzymes tested, (benzyloxycarbonyl)-L-leucyl-L-leucyl-L-ornithinal (2i) showed significant inhibition of kallikrein activity only. Modifications made in the composition and sequence of the P2 and P3 amino acids also resulted in variations in the inhibitory activity of the analogues. In general, plasmin showed a strong preference for inhibitors which contain an L-phenylalanyl-L-leucyl or an L-leucyl-L-valyl unit in the P2 and P3 positions.