For an animal model to predict a compound's potential for treating human disease, inhibitor interactions with the cognate enzymes of separate species must be comparable. Rabbit and human isoforms of stromelysin-1 are highly homologous, yet there are clear and significant compound-specific differences in inhibitor potencies between these two enzymes. Using crystal structures of discordant inhibitors complexed with the human enzyme, we generated a rabbit enzyme homology model that was used to identify two unmatched residues near the active site that could explain the observed disparities. To test these observations, we designed and synthesized three chimeric mutants of the human enzyme containing the single (H224N and L226F) and double (H224N/L226F) mutations. A comparison of inhibitor potencies among the mutant and wild-type enzymes shows that the mutation of a single amino acid in the human enzyme, histidine 224 to asparagine, is sufficient to change the selectivity profile of the mutant to that of the rabbit isoform. These studies emphasize the importance of considering species differences, which can result from even minor protein sequence variations, for the critical enzymes in an animal disease model. Homology modeling provides a tool to identify key differences in isoforms that can significantly affect native enzyme activity.