As part of studies on the interactive effects of light and endocrine factors on hepatic mixed‐function oxidases, the aminopyrine demethylase activity of liver microsomes and the serum concentrations of corticosterone and testosterone were measured in adult male rats (sham‐operated, castrated or adrenalectomized) exposed for 21 days to one of the following types of environmental lighting: (1) Normal light cyle (L from 09.00 to 21.00 h); (2) Reverse light cycle; (3) Constant light (LL) or (4) Constant darkness (DD). One half of the animals in each of the 12 groups was killed at 06.00 h and the other at 18.00 h on the last day of treatment. A 3‐way analysis of variance, multiple comparisons and correlations allowed the following conclusions: (1) The type of lighting had the most significant effect on demethylase activity, its highest values correspondingtoperiods of darkness (particularly in the rats exposed to a reverse light cycle) and its lowest to LL. (2) Castrated rats as a group had the lowest demethylase activity. (3) Time of death was a significant factor, particularly for the rats in a reverse light cycle (higher activity at 18.00 than at 06.00 h). (4) There was a significant and positive correlation between aminopyrine demethylase and the rates of 7α‐hydroxylation of 3β‐hydroxy‐5‐androsten‐17‐one previously reported for the same animals. (5) There was no significant correlation between demethylase activity and serum corticosterone or testosterone.