NADPH-dependent production of acetaminophen-glutathione conjugate has been partly characterized in rat liver microsomes. The reaction showed the characteristics of mixed function oxidase when glutathione concentration was higher than 0.2 mM. It is suggested that hydroxy radical and superoxide are not involved in this reaction. When the reaction was compared among rat, mouse and rabbit microsomes, mouse microsomes showed the highest activity; this was almost 4 times higher than the others, which may, at least in part, explain the susceptibility of mouse to acetaminophen-induced hepatotoxicity. There was also a species difference in the effects of acetone; this was enhanced in the rat microsomes, inhibited in the mouse, and minimally affected in the rabbit.