The genetics of the early interferon response of mouse peritoneal cells to infection with herpes simplex virus type 2 (HSV-2) was studied in susceptible BALB/c and more resistant C57BL/6 mice and in reciprocal crosses between these mice. Wash-outs of the peritoneal cavity of normal C57BL/6 mice contained significantly more cells than wash-outs from BALB/c mice. Therefore, interferon induction with HSV-2 was studied under standardized conditions in vitro. Peritoneal cells reacted to HSV-2 infection by interferon production in a virus dose-dependent manner. Interferon was detected first after 2 h and peaked after 24 h. Cells from C57BL/6 mice of each sex produced significantly more early interferon than cells from BALB/c mice, and cells from female BALB/c mice produced more interferon than cells from males. This difference was not seen with C57BL/6 mice. Cultures of highly purified adherent cells yielded approximately 10 times as much interferon as cultures of non-adherent cells. Since treatment of cells with carbonyl iron and silica significantly reduced the amount of interferon produced, whereas 2000 rad of irradiation had no obvious effect, it is concluded that the main interferon-producing cell in the peritoneal cavity of mice in response to HSV-2 is of the monocyte/macrophage lineage. Interferon production in peritoneal cells was found to be quantitatively influenced by X-linked loci in that cells from male (BALB/c female X C57 male) F1 mice, which inherit the X chromosome from the low-responding BALB/c females, produced significantly lower amounts of interferon than cells from the other three F1 generation genotypes. All interferons were characterized as alpha/beta interferon. It is suggested that the early production of alpha/beta interferon in response to HSV-2 is influenced by X-linked loci, which might be involved in sex-linked differences in resistance to human herpesviruses.