The effect of castration upon hypothalamic LH-RF synthesis and content was determined in mature male rats. In each single experiment 20 hypothalami from both normal and 60-day-castrated rats were bisected into symmetrical portions. For the determination of LH-RF synthesis 20 hypothalamic halves were incubated for 120 min. After incubation the tissue was homogenized in medium containing 1 Nacetic acid, and centrifuged; 1.0 ml of the supernatant was used to test LH-RF activity. In the other 20 halves, hypothalamic LH-RF content was determined: the samples were homogenized without incubation in 1 Nacetic acid, centrifuged, and LH-RF activity was tested in 1.0 ml of the supernatant. LH-RF activity was determined in vivo in ovariectomized rats pre-treated with estrogen-progesterone by measuring, by radioimmunoassay, the LH modifications in serum after the i.v. administration of the extracts. Hypothalamic LH-RF content was significantly higher in control than in castrated rats (p < 0.001). After incubation there were no changes in the LH-releasing potency of control hypothalami compared with non-incubated tissue, whereas a significant increase (616%) was found after the incubation of hypothalami from castrated rats. On the other hand, after incubation LH-RF activity was higher (p < 0.02) in castrated than in control hypothalami. These results suggest that castration stimulates the release and synthesis of hypothalamic LH-RF in male rats.