The purpose of this study was to investigate whether melanin-concentrating hormone (MCH) acts directly on the median eminence and on the anterior pituitary of female rats regulating LHRH and gonadotropin release. In addition, immunohistochemistry was used to examine the density and distribution of MCH-immunoreactive fibers in the median eminence of proestrous rats. MCH-immunoreactive fibers were found in both the internal and external layers of the median eminence and in close association with hypophysial portal vessels. In the first series of in vitro experiments, median eminences and anterior pituitaries were incubated in Krebs-Ringer bicarbonate buffer containing two MCH concentrations (10⁻¹⁰ and 10⁻⁸ M). The lowest MCH concentration (10⁻¹⁰ M) increased (P < 0.01) LHRH release only from proestrous median eminences. Anterior pituitaries incubated with both MCH concentrations also showed that 10⁻¹⁰ M MCH increased gonadotropin release only from proestrous pituitaries. In the second series of experiments, median eminences and pituitaries from proestrous rats were incubated with graded concentrations of MCH. MCH (10⁻¹⁰ and 10⁻⁹ M) increased (P < 0.01) LHRH release from the median eminence, and only 10⁻¹⁰ M MCH increased (P < 0.01) LH and FSH release from the anterior pituitary. The effect of MCH on the stimulation of both gonadotropins from proestrous pituitaries was similar to the effect produced by LHRH. Simultaneous incubation of pituitaries with MCH and LHRH did not modify LH but increased the FSH release induced by LHRH. The present results suggest that MCH could be involved in the regulation of preovulatory gonadotropin secretion.