Follicle-stimulating hormone (FSH), luteinizing hormone (LH) and prolactin concentrations were determined by radioimmunoassay (NIAMD-Rat RIA) of sera from intact hamsters, which were decapitated at selected times during the 4-day estrous cycle. Serum FSH concentrations on Day 1 of the cycle (day of ovulation) were 2- to 6-fold greater than the levels on Day 2 and 5 to 12-fold greater than the mean, “basal,” level of FSH (53 ± 2 ng/ml; 80 hamsters) found during Day 3 and until 1200 hr, Day 4. There was a biphasic serum FSH pattern on Day 4 (proestrus) which was demarcated by the initiation of the preovulatory surge at 1400 hr, the peak level at 1600 hr, the decay of the surge by 2100 hr, and the subsequent elevation to the high levels present on Day 1. In marked contrast to these changes in serum levels of FSH, LH levels were virtually constant throughout the estrous cycle, except for the preovulatory surge between 1300 hr and 1900 hr on Day 4 with a peak level at 1600 hr. Prolactin concentrations varied at most by one-half to 2-fold from each other during diestrus. The preovulatory increase in serum prolactin on proestrus was at a maximum level at 1500 hr, then declined by 2000 hr. (Endocrinology94: 1295, 1974)