Estrogen-2-hydroxylase activity in various rat tissues was surveyed. While the liver, followed by brain, had the highest enzyme activity, nearly all tissues investigated in the male rat possessed 2-hydroxylase activity. Neither the ovary nor the virgin uterus, however, could synthesize 2-hydroxy-estrogens in vitro. Enzyme activities in the male brain and liver were higher than in corresponding female tissues. In the male rat, cytochrome P-450 inducers did not alter brain estrogen-2-hydroxylase activity, but castration caused a fall in enzyme activity in both the brain and liver. Liver estrogen-2-hydroxylase activity was decreased in both hyperthyroid and hypothyroid rats.