Acetaminophen (APAP) is an analgesic and antipyretic agent which may cause hepatotoxicity and nephrotoxicity with overdose in man and laboratory animals.In vivostudies suggest thatin situactivation of APAP contributes to the development of nephrotoxicity. Associated with target organ toxicity is selective arylation of proteins, with a 58-kDa acetaminophen binding protein (58-ABP) being the most prominent cytosolic target. In this study a mouse kidney slice model was developed to further evaluate the contribution ofin situactivation of APAP to the development of nephrotoxicity and to determine the selectivity of protein arylation. Precision cut kidney slices from male CD-1 mice were incubated with selected concentrations of APAP (0–25 mM) for 2 to 24 hr. APAP caused a dose- and time-dependent decrease in nonprotein sulfhydryls (NPSH), ATP content, and K + retention. Preceding toxicity was arylation of cytosolic proteins, the most prominent one being the 58-ABP. The association of 58-ABP arylation with APAP toxicity in this mouse kidney slice model is consistent with earlier,in vivoresults and demonstrates the importance ofin situactivation of APAP for the development of nephrotoxicity. Precision cut renal slices and dynamic organ culture are a good model for further mechanistic studies of APAP-induced renal toxicity..