Interleukin-12 (IL-12) is critical for resistance to Toxoplasma gondii during both the acute and chronic stages of infection. However, the cellular and molecular pathways that regulate IL-12 production during chronic toxoplasmosis are incompletely defined. We recently discovered that 12/15-lipoxygenase (12/15-LOX), which oxidizes unsaturated lipids in macrophages, is a novel and selective regulator of IL-12 production. We now demonstrate the essential role of this enzyme in the chronic phase of toxoplasmosis. Although 12/15-LOX-deficient mice were resistant to acute T. gondii infection, 80% of 12/15-LOX-deficient mice died during chronic toxoplasmosis, compared to no deaths in wild-type controls. The morbidity of chronically infected 12/15-LOX mice was associated with an increase in brain inflammation and parasite burden. These data suggest that the evolution of the immune response to T. gondii is accompanied by an increasing requirement for 12/15-LOX-mediated signaling. Consistent with this conclusion, 12/15-LOX activity was enhanced during chronic, but not acute, toxoplasmosis. Furthermore, the enhanced susceptibility of 12/15-LOX-deficient mice to chronic toxoplasmosis was associated with reduced production of IL-12 and gamma interferon (IFN-gamma) that was not evident during acute infection. Importantly, ex vivo IFN-gamma production by 12/15-LOX-deficient splenocytes could be rescued by the addition of recombinant IL-12. These data establish that 12/15-LOX is a critical mediator of the chronic type 1 inflammatory response and that immune mediators can be subject to distinct cellular and/or molecular mechanisms of regulation at different stages of inflammation.