The compound N-acetyl-p-benzoquinone imine (NAPQI, 1) is a proposed ultimate toxic metabolite in acetaminophen overdose. Previous synthetic methods (e.g., lead tetraacetate oxidation of acetaminophen, dehydration of N-hydroxyacetaminophen, Blair et al.'s stable benzene solutions, Miner and Kissinger's electrochemical generation) did not yield pure NAPQI, which is crucial for confirming its structure and role in acetaminophen toxicity. We describe a method to prepare crystalline NAPQI by oxidizing acetaminophen with freshly prepared silver oxide, followed by silica gel column chromatography and sublimation, resulting in pure product (purity > 99% by HPLC). We investigated the decomposition kinetics of pure NAPQI in buffer using reverse-phase HPLC, finding biphasic disappearance in pH 7.4 sodium phosphate buffer: the second phase exhibited half-order kinetics (rate constant 1.1 × 10^-4 M^(1/2) s^-1, half-life ~11 min), and the initial phase was nearly linear as second order (apparent rate constant 6.25 × 10^-1 M^-1 s^-1); acetaminophen increased the rate of NAPQI disappearance. Preliminary toxicity studies in male BALB/c mice showed NAPQI is significantly more potent (ip LD50 20 mg/kg) than acetaminophen (ip LD50 500 mg/kg), and NAPQI was more cytotoxic to isolated hepatocytes, requiring approximately one-tenth the concentration of acetaminophen for equivalent cell death.