Groups of female rats were decapitated at 3-hr intervals throughout 4-day estrous cycles and the plasma of each rat was assayed for LH, FSH, prolactin, progesterone and estradiol-17β. Radioimmunoassays were used to measure these hormones, except for progesterone which was determined by the competitive protein binding assay. All five hormones exhibited a peak concentration in plasma on the day of proestrus. In addition, progesterone was elevated from 0900 hr of metestrus to 0900 hr of diestrus, while plasma prolactin showed a brief rise at 1500 hr of estrus. The elevated level of estradiol on the day of proestrus followed a different pattern than the other four hormones. Estradiol concentration began to rise late on metestrus, reached a peak at noon of proestrus, and fell before peak levels of the other hormones were reached. The preovulatory elevation of LH, prolactin and progesterone occurred between noon and midnight of proestrus, while the peak of FSH lasted from noon of proestrus to noon of estrus. (Endocrinology94: 1704, 1974)