Uterine natural killer (uNK) cells are transient, large, heavily granulated, maternal lymphocytes present on the mesometrial side of the pregnant mouse uterus. These cells contribute to normal implantation site development. Cytokine production, particularly interferon (IFN)-γ, is a major function of most NK cell subsets. In this study, uNK cells were assessed for IFN-γ production. Local concentrations of IFN-γ were measured in the mesometrial regions of murine implantation sites between Days 6 and 16 of gestation. IFN-γ was detected by ELISA at all days studied in a random-bred (CD1) and an inbred (BALB/c) strain of immune-competent mouse and in two immune-deficient strains, SCID (NK⁺, T⁻, B⁻) and tgϵ26 (NK⁻, T⁻, B⁺). Concentrations of IFN-γ per implantation site peaked at Day 10 of gestation in NK⁺ strains but were low and relatively constant in NK⁻ mice. To evaluate the functions of IFN-γ at murine implantation sites, pregnancy was studied in homozygously mated IFN-γ^−/− and IFN-γRα^−/− mice and their congenic controls. Primiparous but not multiparous IFN-γ^−/− mice experienced significant fetal loss. Primiparous IFN-γRα^−/− carried full litters to term. Implantation site pathology was demonstrated in both strains of gene-deleted mice by light microscopy and ultrastructurally. This included elevated numbers of uNK cells that contained fewer and smaller granules and, after Day 10 of gestation, progressive necrosis and loss of decidua. The presence of a fetus able to produce IFN-γ did not modify the phenotype of pregnant IFN-γ^−/− mice. This study indicates that during murine pregnancy, uNK cells are the main source of IFN-γ on the mesometrial side of the uterus and that IFN-γ contributes to normal health of the midgestational decidua. Furthermore, evidence is presented that IFN-γ-producing cells exist in mesometrial regions of implantation sites that are neither NK nor T cells.