The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces acute nephrotoxicity characterized as polyuric renal failure with proximal tubular necrosis. Phenobarbital pretreatment potentiates NDPS and N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS, a nephrotoxic metabolite of NDPS) nephrotoxicity in male rats. The purpose of this study was to determine the ability of phenobarbital pretreatment to potentiate (1) NDHS nephrotoxicity in female rats and (2) N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA, a nephrotoxic metabolite of NDHS) nephrotoxicity in male and female rats. Age-matched male and female Fischer 344 rats (4/group) were pretreated intraperitoneally (ip) with phenobarbital (75 mg/d, 3 d). At 24 h after the last injection of phenobarbital, an ip injection of NDHS (0.025 mmol/kg), 2-NDHSA (0.025 mmol/kg, females; 0.05 mmol/kg, males), or vehicle was given and renal function was monitored at 24 and 48 h post NDPS metabolite or vehicle. Additional groups received the NDPS metabolite or vehicle only and were also monitored for 48 h. In a separate experiment, male Fischer 344 rats were pretreated with piperonyl butoxide (PIBX, 1360 mg/kg) or the PIBX vehicle. 2-NDHSA (0.1 mmol/kg) or vehicle was administered (ip) 30 min after PIBX, and renal function was monitored for 24 h. Phenobarbital markedly potentiated 2-NDHSA nephrotoxicity in male rats as evidenced by increased kidney weight, increased blood urea nitrogen (BUN) concentration, and decreased tetraethylammonium (TEA) accumulation by renal cortical slices. PIBX had no effect on 2-NDHSA nephrotoxicity. Phenobarbital pretreatment did not markedly enhance the nephrotoxic potential of NDHS or 2-NDHSA in female rats. These results indicate that phenobarbital exhibits differential potentiation of NDPS metabolite nephrotoxicity in male and female rats and that the potentiation of 2-NDHSA nephrotoxicity observed in males is not due to cytochrome P-450-mediated oxidative biotransformation.