4-Aminophenol (PAP) is known to cause nephrotoxicity in the rat where it produces selective necrosis to renal proximal tubules. The aim of this work was to investigate the toxicity of PAP and its known nephrotoxic metabolite 4-amino-3-S-glutathionylphenol using a well defined suspension of rabbit renal proximal tubules. PAP at a concentration of 0.5 mM and 1 mM caused proximal tubule cell death (measured by lactate dehydrogenase release) in a time-dependent manner over a 4-h exposure. In contrast, 4-amino-3-S-glutathionylphenol at 1 mM produced no proximal tubule cell death over a similar 4-h exposure. At 2 h, 1 mM PAP inhibited proximal tubule respiration by 30% and decreased cellular adenosine triphosphate (ATP) levels by 60%. These events preceded cell death. The addition of PAP to proximal tubules led to a rapid depletion of cellular glutathione, exposure to 0.5 mM causing a 50% depletion within 1 h. The cytochrome P-450 inhibitors SKF525A (1 mM) and metyrapone (1 mM), the iron chelator deferoxamine (1 mM) and the antioxidant N,N'-phenyl-1,4-phenylenediamine (2 microM) had no effect on PAP-induced cell death. However ascorbic acid (0.1 mM), afforded a marked protection against the depletion of cellular glutathione and completely protected against the cell death produced by 1 mM-PAP. These results indicate that oxidation of PAP to generate a metabolite that can react with glutathione is an important step in the toxicity, while mitochondria appear to be a critical target for the reactive intermediate formed.