In perfused livers from fed and fasted beta-naphthoflavone-treated C57BL/6J mice, maximal rates of p-nitroanisole O-demethylation were 30-40 mu moles/g/hr and 15-20 mu moles/g/hr respectively. The detergent saponin, at concentrations ranging from 0.001 to 0.005%, was infused between 2 and 30 min to establish optimal conditions to permeabilize plasma membranes. Permeabilization was assessed by release of lactate dehydrogenase and stimulation of p-nitroanisole O-demethylation by citrate. Saponin (0.005% for 5 min) alone had little effect on the rates of p-nitroanisole O-demethylation or conjugation of p-nitrophenol by perfused livers. Further, dicarboxylates or NADPH had no effect on rates of monooxygenation by perfused mouse liver in the absence of saponin. In saponin-treated livers from fasted mice, however, rates of monooxygenation were increased rapidly by infusion of dicarboxylates (10 mM malate, citrate, or isocitrate) or an NADPH-generating system (60 and 110% respectively), over a 6-8 min period. During this time period, cellular energetics were not comprised as reflected by normal rates of glucuronidation of p-nitrophenol. Thus, non-permeable metabolites can enter saponin-permeabilized cells in the perfused liver. Rates of monooxygenation were increased 40-60% in livers from fed mice by citrate, NADPH (200 microM) or an NADPH-generating system. In contrast, saponin decreased mixed-function oxidation assayed in isolated microsomes incubated with an NADPH-generating system. Taken together, these data support the hypothesis that maximal rates of monooxygenation in intact hepatocytes from fed as well as fasted mice is limited by the availability of NADPH.