A system was developed to allow constant monitoring of hepatic cytochrome P450 activity in awake and unrestrained rats. A continuous 13C-aminopyrine perfusion was performed, and breath samples obtained for endogenous CO2 production and 13C measurements, to calculate 13C O2 production due to aminopyrine demthylation. Increasing doses of 13C-aminopyrine produced a hyperbolic increase of expired 13CO2, compatible with an in vivo measurement of enzymatic activity. Acute-cold exposure of the rats during 13C-aminopyrine perfusion produced a two-fold increase of endogenous CO2 production, together with a 27% increased 13C-aminopyrine metabolism (p<0.05 vs basal conditions). In contrast, noradrenaline (20 microg/kg BW/min), despite a similar effect on energy expenditure, did not significantly change 13C-aminopyrine metabolism. Acute-cold exposure is known to stimulate both adrenal catecholamine secretion and the sympathetic nervous system. The observed difference in 13C-aminopyrine demthylation during cold exposure and nonadrenaline perfusion, therefore, could be due to a more specific effect of adrenal catecholamines on liver aminopyrine metabolism. These results suggest the possibility of prolonged in vivo monitoring of liver metabolism pathways such as aminopyrine demethylation, thus allowing the study of drug acute interactions with cytochrome P450 system.