Acetaminophen is an analgesic and antipyretic which causes liver toxicity in humans and experimental animals with overdose. Acetaminophen (APAP) covalent binding to a cytosolic protein of approximately 58 kDa (58-ABP) has been associated with target organ toxicity. Since hepatic content of 58-ABP varies, studies were conducted to determine if this influences APAP binding to other target proteins. In the liver, the amount of 58-ABP varied with individual male CD-1 mice, but in kidneys of the same mice there was no such variability in 58-ABP content. All male A/J mice tested had comparatively little detectable 58-ABP in liver cytosol. Similarly, female CD-1 mice had low 58-ABP content compared to males; however, administration of testosterone propionate to females significantly increased 58-ABP content in liver cytosol. At 4 hr after challenge of mice from the above-described groups with 600 mg APAP/kg, cytosolic covalent binding to proteins was determined by Western blot analysis with anti-APAP antibody. The Western blots were then stripped of antibody and blocking agents and reprobed with antibody prepared against purified 58-ABP (anti-58-ABP). In the liver, the level of APAP bound to the 58-ABP target corresponded with 58-ABP content. In cases where 58-ABP was poorly expressed, APAP adducts to other protein targets were more prominently detected. In the kidneys of the male CD-1 mice 58-ABP arylation by APAP varied little among animals, reflecting the relatively consistent levels of renal 58-ABP. These data suggest that binding to the 58-ABP may spare other potential targets of APAP electrophile attach and support a role of the 58-ABP as a preferred target of APAP electrophile in cytosol.