The present study was designed to test the hypothesis that para-aminophenol (PAP) nephrotoxicity is due to autooxidation. We compared renal functional responses following PAP administration to female Sprague-Dawley rats and following incubation of renal proximal tubules with PAP. The concentrations of PAP selected for in vitro incubations produced cytotoxicity (for example, a decrease in oxygen consumption or adenine nucleotide concentration) in rat renal epithelial cells or rabbit proximal tubule suspensions. In rats, PAP (300 mg/kg i.p.) caused proximal tubular necrosis within 24 hr. Changes in renal function 24 hr following PAP administration included increased kidney weight and blood urea nitrogen concentration and decreased renal glutathione (GSH) content and adenine nucleotide concentrations. PAP did not cause hepatic damage. Within 2-4 hr following PAP administration, renal GSH content and adenine nucleotide concentrations were significantly decreased. In renal cortical slices prepared from PAP-treated rats, oxygen consumption and accumulation of organic ions (para-aminohippurate and tetraethylammonium) were significantly decreased compared with renal cortical slices prepared from control rats. In liver, GSH content was significantly decreased from 1 to 4 hr following PAP administration. In contrast to the effects of PAP in vivo, renal proximal tubules showed little evidence of injury when incubated with 0.1 or 0.5 mM PAP for up to 4 hr in the presence or absence of amino acids in the incubation medium. When tubules were incubated with 1 mM PAP for 4 hr in the presence of amino acids, GSH content, AMP concentration, and TEA uptake were significantly decreased. When amino acids were removed from the incubation medium, 1 mM PAP caused decreases in oxygen consumption and ATP concentration after 4 hr of incubation. Functional changes observed during incubation with PAP in vitro were not consistent with functional changes observed in vivo. The discrepancy between PAP toxicity in vivo and in vitro suggests that autooxidation is unlikely to be responsible for PAP nephrotoxicity and that nephrotoxicity in vivo is primarily mediated by extrarenal bioactivation. Further, depletion of hepatic GSH content prior to changes in renal function suggests that PAP or a PAP metabolite may conjugate with hepatic GSH. These observations suggest that PAP nephrotoxicity may be mediated by PAP-GSH conjugates rather than autooxidation of PAP in the kidney.