The hepatotoxicity of acetaminophen (APAP) is generally attributed to the formation of a reactive quinoneimine metabolite (NAPQI) that depletes glutathione and covalently binds to hepatocellular proteins. To explore the importance of the N-acyl group in APAP metabolism and toxicity, we synthesized 12 acyl side chain homologues of acetaminophen (APAP) and its 3'-regioisomer (AMAP), including the respective N-(4-pentynoyl) analogues PYPAP and PYMAP. Rat hepatocytes converted APAP, AMAP, PYPAP, and PYMAP extensively to O-glucuronide and O-sulfate conjugates in varying proportions, whereas glutathione or cysteine conjugates were observed only for APAP and PYPAP. PYPAP and PYMAP also underwent N-deacylation followed by O-sulfation and/or N-acetylation to a modest extent. The overall rates of metabolism in hepatocytes varied approximately 2-fold in the order APAP < AMAP ˜ PYPAP < PYMAP. Rat liver microsomes supplemented with NADPH and GSH converted APAP and PYPAP to their respective glutathione conjugates (formed via a reactive quinoneimine intermediate). With PYPAP only, a hydroxylated GSH conjugate was also observed. Thus, differences in biotransformation among these analogues were modest and mostly quantitative in nature. Cytotoxicity was evaluated in cultured hepatocytes by monitoring cell death using time-lapse photomicrography coupled with Hoechst 33342 and CellTox Green dyes to facilitate counting live cells vs dead cells, respectively. Progress curves for cell death and the areas under those curves showed that toxicity was markedly dependent on compound, concentration, and time. AMAP was essentially equipotent with APAP. Homologating the acyl side chain from C-2 to C-5 led to progressive increases in toxicity up to 80-fold in the para series. In conclusion, whereas N- or ring-substitution on APAP decrease metabolism and toxicity, homologating the N-acyl side chain increases metabolism about 2-fold, preserves the chemical reactivity of quinoneimine metabolites, and increases toxicity by up to 80-fold.