Epidemiological studies have shown that smoking during pregnancy markedly increases the risk for future tobacco use by adolescent female offspring. It has been hypothesized that the increased smoking risk in females is secondary to a nicotine-induced increase in fetal plasma testosterone levels that persist to adult life. To test this hypothesis, we randomized pregnant Sprague-Dawley rats to receive either saline or nicotine from Day 4 until the end of gestation. Blood samples for testosterone levels were obtained from 30- and 120-day-old offspring. In addition, blood samples for testosterone levels were obtained prior to and following a 2-day infusion of nicotine to chronically catheterized ovine fetuses. Maternal nicotine exposure resulted in increased plasma testosterone in 30-day-old female rat offspring, with no differences found in nicotine-exposed males. In addition, plasma testosterone levels increased in ovine fetuses in response to the nicotine infusion. We conclude that prenatal nicotine exposure increases plasma testosterone levels chronically in adolescent female rat offspring and acutely in both male and female ovine fetuses. Although our findings lack correlative behavioral information on the female offspring, these data are consistent with human epidemiological data suggesting that prenatal environmental influences may have marked effects on the subsequent smoking behaviors of offspring.