Human leukocytes produced interferon-gamma (IFN-gamma) rapidly without induction during incubation at 37 degrees C. The intracellular level peaked at 3 h; the extracellular IFN-gamma reached its maximum at 7 h. Leukocytes purified by different methods, and whether from single donors or pooled from numerous donors, yielded similar amounts of IFN. The best yields were approximately 3 IU/10(6) cells. The highest titers were obtained at cell concentrations ranging between 30 and 100 X 10(6)/ml, and at these concentrations, the production of IFN-gamma was not affected by the presence or absence of serum. At lower cell concentrations, the yield of IFN per cell decreased markedly in the absence of serum. The cultural conditions and medium were not very critical; leukocytes incubated in various suspension and stationary cultures consistently produced IFN-gamma. We did not identify the mechanism responsible for the rapid production of IFN-gamma in vitro, but found no evidence of a role of calcium. Immunoaffinity-purified IFN-gamma from uninduced leukocytes and from leukocytes induced with lentil lectin behaved differently in gel filtration.