Abstract The influence of p-nitroanisole, a substrate for mixed-function oxidation, on total NADP &+ and NADPH and NADP +/NADPH ratios was examined in perfused livers from three different species. Studies were performed using livers from Sprague-Dawley rats, Syrian golden hamsters and C57BL/6J mice. Although rates of p-nitroanisole O-demethylation varied more than 16-fold in perfused livers from these species, NADP +/NADPH ratios calculated from measured concentrations of NADP + and NADPH and from ratios calculated from substrate pairs assumed to be in near equilibrium with NADP +-dependent dehydrogenases remained remarkably constant under most conditions. Thus, rates of NADPH utilization and generation must be tightly coupled in perfused livers during high rates of mixed-function oxidation. Exceptions to the general pattern noted above occurred in livers of fasted, phenobarbital-treated rats where carbohydrate reserves were depleted and in livers from 3-methyl-cholanthrene-treated mice where rates of p-nitroanisole O-demethylation were exceptionally high. Livers from fed phenobarbital-treated rats displayed a paradoxical decrease in NADP +/NADPH ratios reflecting reduction calculated from substrates assumed to be in near equilibrium with 6-phosphogluconate dehydrogenase during mixed-function oxidation, suggesting that NADPH generation exceeded NADPH utilization in the rat in the fed state. In contrast, the NADP +/NADPH ratio calculated from measured pyridine nucleotides increased in livers of 3-methylcholanthrene-treated mice perfused with p-nitroanisole, reflecting oxidation. Moreover, the NADP +/NADPH ratio calculated from substrates assumed to be near equilibrium with 6-phosphogluconate dehydrogenase increased in livers of fasted rats, suggesting that utilization of NADPH exceeded generation. Thus, adequate carbohydrate reserves appear essential for maintenance of NADPH during high rates of mixed-function oxidation.