To define the major cell populations contributing to IFN-␥ production, peritoneal excudate cells (PECs) from uninfected (0 h) mice and mice infected with LCMV for 20, 24, 30, 36, 40 , and 48 h were isolated, stained for NK1.1 and T cell receptor ␤ (TCR-␤), fixed, and permeabilized to stain for cytoplasmic IFN-␥ (see Materials and Methods for staining and gating strategies). Total lymphocytes were evaluated with an extended lymphocyte gate. The NK cell subpopulations were identified as NK1.1 ϩ TCR-␤ Ϫ and the T cell subpopulations as NK1.1 Ϫ TCR-␤ ϩ . Lymphocytes isolated from uninfected mice, including total, NK, and T cells, did not basally express detectable IFN-␥; the positive frequencies for all populations were Ͻ4% ( Fig. 1B and C) . By 24 h after challenge with LCMV, approximately 14 to 37% of the NK cells expressed moderate to high levels of intracellular IFN-␥. The peak of this NK cell response was at 30 h after infections, when 28 to 43% of the NK cells expressed IFN-␥. The response was resolving at 36 to 48 h. In contrast to the NK cells, neither total lymphocyte nor T cell populations had high IFN-␥ levels at any of the times examined ( Fig. 1B and C) . These results demonstrate that during LCMV infection, peritoneal NK cells respond with IFN-␥ expression and are the major producers of the cytokine in the peritoneal cavity.