Nocardia asteroides causes an acute, necrotizing pneumonia characterized by extensive infiltration of polymorphonuclear neutrophils (PMNs) into the lungs. Although PMNs have historically been classified as end-point cells, recent investigations have indicated that PMNs have the ability to secrete cytokines such as interleukin (IL)-4 and IL-12. This study investigated the ability of PMNs to produce cytokines in a murine model of N. asteroides pulmonary infection. Flow cytometric analysis demonstrated the production of interferon-gamma (IFN-gamma), but not IL-4, by PMNs in response to this infection. IFN-gamma production correlated with peak infiltration of PMNs into the lungs. Cell sorting and enzyme-linked immunosorbent assay were used to confirm cytokine production by cells with nuclear morphology characteristic of PMNs. This is the first report of IFN-gamma production by neutrophils in response to an infection in vivo. These results suggest that PMNs play an important role in directing the host toward a T helper cell type 1 phenotypic response in the lungs.