The effect of photoperiod and reproductive cycle on the fluctuations in basal serum cortisol (F) levels were determined in adult female guinea pigs bled by intracardiac puncture under light methoxyflurane anesthesia. Blood samples were collected at regular intervals over a 24 hour period in order to assess the effects of the lighting regime (14 hr light/day: lights on 0600 hr) on basal levels of F. Peak F levels were observed at the beginning of the light cycle and lowest levels observed just prior to the onset of darkness in random, cyclic female guinea pigs. When analyzed for the influence of reproductive state, basal F levels were found to be elevated during proestrus, Day 2 (Day 0 = estrus) and the mid-luteal (Day 6-7) period of the estrus cycle. In ovariectomized animals a 10 microgram dose of estradiol (E) induced a dramatic rise (P less than 0.001) in adrenal weight and the basal levels of serum F as compared with oil-treated controls. Lower doses of E (1 microgram) and combined progesterone (2 mg)-E (1 microgram) treatment induced an elevation in adrenal weight and/or basal levels of serum F. Progesterone administration alone had no effect on either of these adrenal indices as compared with controls. These data indicate that both photoperiod and ovarian steroid hormone milieu affect adrenal function in the female guinea pig.