Abstract Strain differences in susceptibility of rats to acetaminophen (APAP)-induced nephrotoxicity have been previously reported. Young adult male Fischer-344 (F-344) rats are susceptible whereas weight-matched Sprauge-Dawley (SD) rats are not susceptible to APAP nephrotoxicity. The present study was designed to evaluate the role of pharmacokinetics in strain-dependent APAP nephrotoxicity. Age-matched (2-month-old) male F-344 and SD rats received 250–750 mg APAP/kg, i.v., or 0–1000 mg APAP/kg, i.p. Pharmacokinetic variables were evaluated following i.v. APAP and 24 h urinary excretion of APAP and major metabolites was determined following both i.v. and i.p. administration of APAP. Following i.p. administration, nephrotoxicity was observed only in F-344 rats following 1000 mg APAP/kg; SD rats were not susceptible to APAP-induced nephrotoxicity. In contrast, nephrotoxicity did not occur in either F-344 or SD rats administered APAP i.v. Pharmacokinetic variables (volume of distribution, apparent systemic clearance, and apparent terminal half-life) of APAP were similar in F-344 and SD rats. No striking differences in the pattern of specific urinary metabolites were observed between F-344 and SD rats treated with i.p. or i.v. APAP. Thus, strain differences in APAP-induced nephrotoxicity do not appear to be due to differences in pharmacokinetics or major pathways of APAP metabolism.