Successful immunity to Toxoplasma gondii requires a strong cell-mediated immune response. Neutrophils possess the ability to rapidly migrate into tissues in response to microbial stimuli. Therefore, we sought to determine whether murine neutrophils could respond to T. gondii by producing immunoregulatory cytokines. We show that murine neutrophils produce high levels of IL-12 and low, but significant, levels of TNF-alpha when stimulated with T. gondii Ag. Both cytokines are produced in the absence of IFN-gamma. Production of IL-12 does not require TNFR p55, and release of TNF-alpha occurs independently of IL-12. We show that there is an influx of neutrophils into the peritoneal cavity that peaks at approximately 8 h in response to injection of live tachyzoites and that this is correlated with increased transcription of IL-12 p40. Our results establish that murine neutrophils possess the ability to produce immunoregulatory cytokines during T. gondii infection and suggest that this response may be important in early host defense and in triggering cell-mediated immunity to the parasite.