Lymphocyte products released during the human mixed reaction were studied for their ability to stimulate human monocytes to produce endogenous pyrogen and lymphocyte activating factor. These two biological activities are considered properties of the same molecule, called interleukin-1 (IL-1). In these experiments, physical characteristics such as molecular weight, isoelectric point, and binding to concanavalin A (Con A) sepharose were studied under conditions which excluded bacterial endotoxins. Gel filtration revealed molecular weights of approximately 60 and 25 kD with IL-1-inducing activity. Isoelectric points ranged from 5.9 to 6.3. The IL-1-inducing properties of mixed leukocyte reaction supernates did not bind to Con A sepharose. Recombinant human gamma interferon did not induce IL-1 production under various conditions but rather augmented IL-1 induced by endotoxin. In contrast, the mixed leukocyte reaction results in production of lymphokines which directly stimulate IL-1 production in the absence of endotoxins.