Standard methods for studying the maturation of the hepatic monooxygenase system in neonatal rats require killing groups of animals at various ages. We have developed a simple noninvasive technique which can be used in serial studies on a single animal over a period of time and which accurately reflects changes in hepatic aminopyrine (AP) N-demethylase activity. Rats between the ages of 2 hr and 21 days were injected ip with 4-(N,N-di[14C]methyl)aminoantipyrine and the expired 14CO2 was continuously collected over a period of 3 hr. The peak rate of expired 14CO2 ranged from 0.0054% of the dose per min in 2-hr-old neonates to 0.28% of the dose per min in 21-day-old rats. In order to validate the AP CO2 breath test (ABT), individual mean rates of expired 14CO2 at 25 min and hepatic 9000g supernatant AP N-demethylase activity were compared in the developing neonate between the ages of 2 hr and 21 days. the correlation between changes in mean rate in vivo (as determined by the ABT) and 9000g supernatant AP N-demethylase activity per liver throughout development was excellent (r2 = 0.94). Finally, differences in the ABT of male and female rats were found to reflect expected changes in hepatic AP N-demethylase activity in vitro.