In a previous study, a 65 kDa protein, TDI-65, was found to be accumulated in the leaves of drought-stressed tomato (Lycopersicon esculentum cv. Starfire) plants. The protein level returns to control level when the drought-stressed plants are rewatered. Antibodies raised against the purified protein were used to elucidate the subcellular localization of the protein. The protein was found to be mainly localized in the nuclei and chloroplasts of drought-stressed leaf cells. To identify the nature of the protein, a cDNA library was constructed and screened by the purified anti-TDI-65 antibody. A cDNA clone designated tdi-65 was isolated and characterized. The deduced amino acid sequences of tdi-65 protein has extensive homology with known cysteine proteases such as actinidin and papain. Northern blot analysis revealed that tdi-65 mRNA is 10-fold higher in drought-stressed plants as compared to control and rewatered plants. Similar results were observed in the tomato cultivar Ailsa and its near isogenic abscisic acid (ABA)-deficient mutant line, flacca, suggesting that the gene does not require ABA for its expression under drought conditions. Based on the previous immunolocalization findings we suggest that tdi-65 encoded cysteine protease functions in relation to drought-induced senescence and programmed cell death.