Homogenates of male rat hypothalami were fractionated by differential centrifugation. Of the total quantity of TRH and LRH in the homogenate, about 50% was in particles sedimenting at 11,500 X g. and 15-20% was in particles sedimenting between 20,000 and 105,000 X g. No LRH or TRH was recovered in the 105,000 X g supernatant fluid. When the 900 X g supernatant fluid was subjected to continuous sucrose density gradient centrifugation, TRH-containing particles distributed as two peaks located at 0.9 and 1.1 M sucrose. On the other hand, LRH-containing particles distributed as only one peak located at 1.2 M sucrose. The 11,500 X g pellet contained those particles comprising the 1.2 M peak of LRH and the 1.1 M peak of TRH. Acid phosphatase activity was found in the gradient fractions containing TRH and LRH, whereas NADPH-cytochrome c reductase and cytochrome c oxidase activities were separated from the peaks of TRH and LRH. Norepinephrine, dopamine, and TRH distributed identically on the gradient. Hypo-osmotic treatment changed the gradient profile of TRH but not that of LRH. Most of the TRH was found near the top of the gradient, but a small amount of TRH was associated with particles which were lighter than those previously noted. The peak of LRH was reduced but its location on the gradient was unchanged. It is concluded that in hypothalamic homogenates TRH and LRH are localized in synaptosome-like particles which have different physical properties.