The mechanism of cinnamaldehyde (CA) cytotoxicity has been investigated in isolated F344 rat hepatocytes. CA reacts spontaneously with reduced glutathione (GSH) in the test-tube. In contrast, cinnamic acid (CAC) and cinnamyl alcohol (CALC) do not react with GSH. CA is the reactive cinnamyl species, since this compound is the most potent intracellular GSH depletor and exhibits the highest cytotoxicity in hepatocyte suspensions. The dose-response curve for CA is very steep, with a threshold cytotoxic concentration of 10(-3)m. CALC is equipotent with CA in depleting GSH but exhibits a latency in this effect, consistent with CALC being metabolized to CA in order to deplete GSH. CAC had no effect on GSH and was non-cytotoxic. Predepletion of GSH with diethyl maleate and/or inhibition of gamma-glutamylcysteine synthetase by l-buthionine S,R-sulfoximine reduces the threshold concentration for cytotoxicity. Inhibition of oxidation of CA to CAC (the major route of metabolism) with cyanamide also increases both CA cytotoxicity and GSH depletion. Ethanol increases GSH depletion by CA without inducing cytotoxicity, but has no further effect on the GSH depletion or cytotoxicity in cyanamide and CA-treated suspensions.