The exhalation of 14CO2 after the administration of [dimethylamino-14C]aminopyrine to an organism is assumed to reflect the demethylation of aminopyrine by hepatic mixed-function oxidase activity. The formaldehyde formed as a result of the demethylation of aminopyrine is then sequentially oxidized to formic acid and CO2. The last step in the pathway, i.e., formate oxidation, is dependent upon tetrahydrofolate; thus, factors which alter hepatic tetrahydrofolate potentially may modify 14C-aminopyrine metabolism to 14CO2in vivo. Exposure of rats to nitrous oxide (N2O) produces a significant reduction in hepatic tetrahydrofolate as a result of the inhibition of 5-methyltetrahydrofolate:homocysteine methyltransferase activity (E.C. 188.8.131.52). In the present study, exposure of rats to N2O/O2 (1:1) for 4 hr prior to the administration of 14C-aminopyrine (40 or 400 Mmoles per kg) produced a 60% reduction in the peak rate of 14CO2 exhalation and a 45% decrease in the total 14CO2 exhaled within 2 hr. In control experiments, exposure of rats to nitrogen/O2 (1:1) produced no effect on 14C-aminopyrine metabolism to 14CO2. Administration of methionine (1.3 mmoles per kg) 30 min prior to 14C-aminopyrine administration reversed the inhibition of 14CO2 exhalation and reduction in hepatic tetrahydrofolate observed in N2O-exposed animals. Aminopyrine (400 pmoles per kg) administration to air-breathing rats did not affect the level of urinary formate, but exposure to N2O produced a 40-fold increase. Aminopyrine administration to N2O-exposed rats produced a 75% increase in urinary formate as compared to rats treated with N2O alone. The first-order rate constant for aminopyrine elimination from the plasma, apparent volume of distribution, apparent plasma clearance and the level of hepatic cytochrome P-450 were not affected by N2O exposure. N2O exposure inhibited 14CO2 exhalation after [N-methyl-14C]morphine administration to a similar degree as that observed after aminopyrine administration. These results indicate that factors which alter the level of hepatic tetrahydrofolate, e.g., nutritional status, N20 exposure or certain drugs, may modify the metabolism of 14C-amino-pyrine to 14CO2in vivo without affecting the demethylation of aminopyrine. Thus, such factors must be controlled when the exhalation of 14CO2 after 14C-aminopyrine administration to an organism is used as an in vivo measure of hepatic drug metabolism.