We report the isolation from tomato (Lycopersicon esculentum) of an ethylene-responsive member of the proteinase inhibitor gene family. DNA sequence analysis of a full-length cDNA clone indicates that the ethylene-responsive gene is distantly related to the tomato proteinase inhibitor I gene, having 53% sequence identity. The predicted amino acid sequence reveals 47% and 45% sequence identity with the tomato and potato proteinase inhibitor I polypeptides, respectively. Additionally, the ethylene-responsive inhibitor has evolved a completely different pattern of gene expression and inhibitory specificity than other members of the inhibitor I family. Gel blot hybridization experiments show that, unlike the tomato proteinase inhibitor I gene, it is not induced in wounded leaves. In contrast, it is activated by the plant hormone ethylene in leaves and during fruit ripening. Furthermore, the ethylene-responsive inhibitor exhibits a novel reactive site, having glutamic acid as the P1 residue. This suggests that the ethylene-responsive proteinase inhibitor does not react with chymotrypsin, as does proteinase inhibitor I, but that it reacts with proteolytic enzymes that cleave at glutamic residues, such as the Staphylococcus aureus V8 proteinase, for which no inhibitors are known. Finally, isolation and analysis of a genomic clone reveals that the ethylene-responsive proteinase inhibitor gene is tightly linked to another, yet unidentified, coordinately expressed gene. We discuss these results with regard to the function and evolution of proteinase inhibitor genes in tomato.