Lysates of human leukocytes, prepared by ultrasonic disruption, contained heat-labile histamine-releasing factors. An inhibitor of histamine release was generated from such lysates during thermal inactivation of the histamine-releasing activity. Lysates inactivated at 38 degrees C for 60 min produced up to 90% inhibition of histamine release from human leukocytes. Heated lysates produced significant inhibition (P < 0.001) of leukocyte histamine release induced by goat antihuman IgE (51+/-3% inhibition), compound 48/80 (69+/-7% inhibition), and fresh leukocyte lysate (63+/-6% inhibition). Membrane ultrafiltration and gel-filtration chromatography suggested that the inhibitor was of less than 1,000 molecular weight. The partially purified inhibitor, eluted from Sephadex G-15 and lyophilized, contained no detectable protein or carbohydrate but produced 80-90% inhibition of histamine release at a concentration of only 10 ng/ml. The histamine-releasing factors from lysates of purified suspensions of lymphocytes and granulocytes were fractionated on Sephadex G-25. A single peak of histamine-releasing activity with an estimated molecular weight of 5,000 was detected from each cell type. Incubation of the partially purified histamine-releasing factors at 38 degrees C resulted in generation of an inhibitor of histamine release which was nearly identical to the inhibitor from crude lysates. This natural inhibitor of histamine release, derived from human leukocytes, may provide an endogenous mechanism for the control of histamine release in inflammation.