Cultures of peritoneal exudate cells (PEC) of non-immune C57BL/6 mice reacted to infectious herpes simplex virus type 1 (HSV), but not to non-infectious virions by producing interferon. Similarly, when the virus preparations were injected intraperitoneally (i.p.) and interferon was determined in the wash-out fluid, interferon could only be detected after injection of infectious HSV. All interferons were mixtures of interferon-alpha and -beta. The cell that produced interferon was not sensitive to anti-theta serum or anti-asialo-GM1 serum, it was retained on nylon wool columns and removed by plastic adherence. Silica treatment of PEC abolished HSV-induced interferon production whereas irradiation by 2000 R was without effect. Pure cultures of mouse T cells did not produce interferon when treated with infectious HSV whereas pure cultures of mouse bone marrow macrophages did produce interferon. Thus, we suggest that in PEC the cells which produced interferon were of the macrophage lineage. In further experiments we have analysed in vivo activation of natural killer (NK) cells in the peritoneal cavity of mice. Non-infectious virions were as effective as infectious HSV and doses of HSV too low to induce measurable titres of interferon caused a marked activation of NK cells.