The human homologue of the Drosophila tumor suppressor lethal (2) tumorous imaginal discs (l(2)tid) gene, hTID1, encodes two proteins derived from alternate mRNA splicing. The splice variants TidL and TidS were previously reported from protein overexpression and dominant-negative mutant protein studies to exhibit opposing biological activities in response to exogenous cytotoxic stimuli. TidL was found to promote apoptosis while TidS suppressed it. To elucidate the physiological function of hTID1, we depleted hTID1 proteins using the technique of RNA interference (RNAi). Here, we show that cells essentially lacking expression of hTID1 proteins are protected from cell death in response to multiple stimuli, including cisplatin, tumor necrosis factor alpha/cycloheximide and mitomycin C. We also generated stable cell populations depleted of hTID1 proteins by RNAi using DNA vectors. In addition to apoptosis resistance, stable hTID1 knockdown cells exhibited an enhanced ability for anchorage-independent growth, as measured by an increase in soft-agar colony formation. These results suggest that hTID1 functions as an important cell death regulator and raise the interesting possibility that hTID1 could exert tumor suppressor activity.