Administration of caffeine, ip 100 mg/kg/day for 1-5 days, to adult male rats resulted in a significant increase in hepatic cytochrome P-450 and b5 concentrations and in cytochrome c reductase, aminopyrine N-demethylase and acetanilide hydroxylase activities. No change was seen in relative liver weight but microsomal protein content was increased after treatment for 1 day and decreased after treatment for 3 or 5 days. In adult rats given 25, 100 or 150 mg caffeine/kg for 3 days, maximum stimulation of mixed-function oxidases was seen with the 100-mg/kg dose. Caffeine treatment (100 mg/kg for 3 days) increased relative liver weight in female guinea-pigs and decreased it in chicks and female mice, and decreased microsomal protein content in male mice, female guinea-pigs and young rats, and increased it in chicks. A significant increase in hepatic cytochrome P-450 content was seen in all species studied. Cytochrome b5 content was increased in chicks and young rats, while cytochrome c reductase activity was increased in male and female mice, young rats and chicks and decreased in female guinea-pigs. Aminopyrine N-demethylase activity was increased in young rats and female guinea-pigs, and acetanilide hydroxylase was increased in all test species except male mice. In vitro addition of 2.5 mM-caffeine to microsomal incubations from untreated rats, guinea-pigs, mice and chicks inhibited aminopyrine N-demethylase activity, although only to a significant extent in male mice; addition of caffeine to incubations containing microsomes from caffeine-treated animals produced significant inhibition of aminopyrine N-demethylase activity in microsomes from adult and young rats and female guinea-pigs. Aminopyrine N-demethylase inhibition did not increase with increasing concentration of added caffeine, although acetanilide hydroxylase activity was progressively inhibited in the microsomal incubates from both control and caffeine-treated animals.