A mitochondrial fraction prepared from homogenates of rat hypothalamic tissue was found by means of electron microscopy to be enriched with synaptosomes. The release of luteinizing hormone releasing hormone (LHRH) and thyrotropin releasing hormone (TRH) from this preparation was investigated. After incubation, the synaptosomes were re-isolated by ultrafiltration; and the concentration of LHRH and TRH in the ultrafiltrate was determined by radioimmunoassay. When the synaptosome-enriched preparation was incubated in 0.32M sucrose at 1 or 30 C, less than 10% of the total LHRH and TRH was recovered in the ultrafiltrate. The two hormones were released by depolarizing concentrations (60 mM) of K+ in a Ca++-dependent manner, and the stimulatory effect of K+ was essentially complete within 2 min. In the presence of 2 mM Ca++, the release of LHRH and TRH increased with increasing K+ concentrations in the range 30-120 mM. Prostaglandin E2 (PGE2), PGF2 alpha, and PGF2 beta had little if any effect on LHRH or TRH release. When the synaptosome-enriched fraction was incubated in Hanks' balanced salt solution, the release of LHRH and TRH was about 10 times greater than that seen in 0.32M sucrose. It is concluded that a synaptosome-enriched fraction from the hypothalamus contains readily releasable pools of LHRH and TRH which are mobilized rapidly by depolarizing concentrations of K+ in a Ca++-dependent manner.