The human squamous cell carcinoma antigens (SCCA) 1 and 2 are members of the serpin family that are 92% identical in their amino acid sequence. Despite this similarity, they inhibit distinct classes of proteinases. SCCA1 neutralizes the papain-like cysteine proteinases, cathepsins (cat) S, L, and K; and SCCA2 inhibits the chymotrypsin-like serine proteinases, catG and human mast cell chymase. SCCA2 also can inhibit catS, as well as other papain-like cysteine proteinases, albeit at a rate 50-fold less than that of SCCA1. Analysis of the mechanism of inhibition by SCCA1 revealed that the reactive site loop (RSL) is important for cysteine proteinase inhibition. The inhibition of cats by a mutant SCCA2 containing the RSL of SCCA1 is comparable to that of wild-type SCCA1. This finding suggested that there were no motifs outside and only eight residues within the RSL that were directing catS-specific inhibition. The purpose of this study was to determine which of these residues might account for the marked difference in the ability of SCCA1 and SCCA2 to inhibit papain-like cysteine proteinases. SCCA2 molecules containing different RSL mutations showed that no single amino acid substitution could convert SCCA2 into a more potent cysteine proteinase inhibitor. Rather, different combinations of mutations led to incremental increases in cats inhibitory activity with residues in four positions (P1, P3', P4', and P11') accounting for 80% of the difference in activity between SCCA1 and SCCA2. Interestingly, the RSL cleavage site differed between wild- type SCCA2 and this mutant. Moreover, these data established the importance of a Pro residue in the P3' position for efficient inhibition of cats by both wild-type SCCA1 and mutated SCCA2. Molecular modeling studies suggested that this residue might facilitate positioning of the RSL within the active site of the cysteine proteinase.