Of antifoulants that are substitutes for organotin compounds such as tributyltin and triphenyltin, N-(2,4,6-trichlorophenyl)maleimide (IT-354) is listed as a much less toxic agent, although the available information concerning IT-354 toxicity is the results of acute toxicity tests in freshwater fish. In this study, the effects of IT-354 on rat thymic lymphocytes were examined using flow-cytometric techniques with appropriate fluorescent probes in order to estimate the effects of IT-354 on mammalian cells. Treatment of cells with 1-10 μM IT-354 for 1 hr did not increase the population of dead cells (cell lethality). However, 10 μM IT-354 significantly increased the population of living, annexin V-positive cells. Annexin V-positive, living cells are expected to be undergoing apoptosis. IT-354 at 3-10 μM significantly elevated intracellular Ca²⁺ and Zn²⁺ levels mainly by increasing Ca²⁺ influx and intracellular Zn²⁺ release. Furthermore, IT-354 significantly depolarized membranes and decreased cellular non-protein thiol content. Assessments using selected antifouling agents showed that the cellular actions of IT-354 are most likely similar to those of other commonly used antifouling agents. Therefore, the toxic potency of IT-354 on wild mammals is speculated to be similar to those of the other tested antifoulants.