The spontaneous and glutathione (GSH) transferase catalyzed reactions of GSH and N-acetyl-p-benzoquinonimine (NABQI) have been studied by stopped-flow kinetics. The spontaneous reaction was shown to be first order in NABQI, GSH and inversely proportional to the H+ concentration; e.g., at pH 7.0 and 25 degrees C the second-order rate constant was 3.2 X 10(4) M-1 s-1. Data for the enzymatic reaction gave values for Km of 27, 1.3, 7, and 7 microM and values for kappa cat of 90, 37, 5.1, and 165 s-1 for rat liver GSH transferases 1-1, 2-2, 3-3, and 7-7, respectively. Over a wide range of reactant concentrations and pH, the spontaneous reaction yields three products, namely a GSH conjugate, 3-(glutathion-S-yl)acetaminophen; a reduction product, acetaminophen; and an oxidation product, glutathione disulfide in the proportions 2:1:1. Analysis of products formed after enzymatic reaction showed that both GSH conjugation and the reduction of NABQI to acetaminophen were catalyzed to an extent characteristic of each isoenzyme. With respect to GSH conjugation, GSH transferase isoenzymes were effective in the order 7-7 greater than 2-2 greater than 1-1 greater than 3-3 greater than 4-4, and with respect to NABQI reduction these isoenzymes were effective in the order 1-1 greater than 2-2 greater than 7-7 the position of isoenzymes 3-3 and 4-4 being uncertain. Human GSH transferases delta, mu, and pi behave similarly to the homologous rat enzymes, i.e., toward conjugation in the order pi greater than delta greater than mu and the reduction delta greater than mu greater than pi (for nomenclature see W. B. Jakoby, B. Ketterer, and B. Mannervik, (1984) Biochem. Pharmacol. 33, 2539-2540). Possible mechanisms of the reaction and its effect on the toxicity of NABQI are discussed.