Interferon gamma (IFNγ) has been implicated as a mediator of luteal steroidogenesis and cell fate. IFNγ-initiated signaling events, although implied by studies in cell lines, have yet to be described in primary luteal cells. The objective of these studies was to begin to characterize IFNγ-initiated signaling within luteal cells. Dispersed bovine luteal cell cultures were challenged with increasing levels of bovine recombinant IFNγ (0–1000 U) or IFNγ (200 U) in the presence or absence of tumor necrosis factor α (TNFα, 10 ng/ml) over time (short term, 0–60 min; long term, 0, 24, 48 h). Fractionated or total cell lysates were evaluated by the Western blotting technique to determine the changes in the levels of signal transducers and activators of transcription (STAT), interferon regulatory factor 1 (IRF-1), and I kappa B α (IκB-α). Utilizing antibodies that recognize the nonphosphorylated forms of STAT-1 and STAT-3, it was determined that levels of STAT-1 and STAT-3 in total cell lysates were constitutively expressed and did not change in response to treatment with IFNγ or TNFα. In contrast, nuclear levels of STAT-1 and phosphorylated STAT-3 were elevated in a time-dependent manner in response to IFNγ treatment. Furthermore, IFNγ and TNFα treatment elevated levels of IRF-1 within 2 h. TNFα-induced increases in the levels of IRF-1 were transient, whereas the levels of IRF-1 in response to IFNγ treatment remained elevated at 48 h. These data suggest that IFNγ treatment can activate members of the STAT pathway, resulting in increased levels of IRF-1. TNFα treatment induced a rapid decrease in the [bu791]levels of IκB-α. IFNγ treatment did not alter the levels of IκB-α and failed to inhibit the TNFα-initiated decrease in the levels of IκB-α. The present experiment demonstrates that the steroidogenic cells of the corpus luteum have the capacity to respond to IFNγ via activation of STAT and IRF-1, providing further evidence that IFNγ may be involved in the luteolytic process. These data also suggest that IFNγ does not signal through the nuclear factor κ B cell survival signaling pathway.