Relative cytotoxicity and mechanism of action of thiophenes were investigated in suspensions of freshly isolated renal proximal tubular (PT) and distal tubular (DT) cells using 4-(2-thienyl)butyric acid (TByA) as a model compound. TByA produced significantly greater time- and concentration-dependent cytotoxicity, as assessed by release of lactate dehydrogenase, in PT cells than in DT cells. Greater intracellular accumulation of TByA in PT cells may play a role in the enhanced cytotoxicity. Pretreatment of cells with alpha-tocopherol partially protected both PT and DT cells from TByA-induced cytotoxicity, suggesting that oxidative stress may be involved in the mechanism of action. In contrast, pretreatment of cells with SKF-525A, an inhibitor of cytochrome P-450, enhanced TByA-induced cytotoxicity in both cell populations, particularly in DT cells. Enhanced accumulation of TByA in SKF-525A-pretreated cells and additional bioactivation mechanisms may be responsible for the enhancement of cytotoxicity. TByA produced GSH oxidation, lipid peroxidation, and inhibition of cellular respiration in both cell populations, supporting involvement of oxidative stress and mitochondrial dysfunction in cytotoxicity.