Supernatants from 1- to 2-day cultures of human mononuclear cells induced the release of histamine from basophils. Generation of this histamine-releasing activity (HRA) was stimulated by addition of concanavalin A to the cell cultures. Mononuclear cells were also cultured with SKSD and Candida albicans antigens. Stimulation of HRA production by these antigens was correlated with positive delayed skin reactions. Serial dilutions of supernatants assayed for HRA provided a semiquantitative determination of the level of HRA in mitogen- or antigen-stimulated samples. Antigen increased HRA production when added during the first or second day of culture. Generation of HRA probably requires active protein synthesis, since puromycin was inhibitory, and since preformed HRA could not be recovered from lysed cells. HRA was detected in supernatants after 4 hr, and the effects of antigen stimulation were apparent after 8 hr of culture. The rate of HRA synthesis diminished after 18 hr of culture. Replacement of supernatants with fresh culture medium allowed continued synthesis of substantial quantities of HRA during the second day of culture. A linear correlation was observed between the amount of HRA produced and the mononuclear cell concentration. Our findings provide evidence for the interaction of lymphocytes and basophils via a soluble mediator.