The cellular and cytokine responses in the lungs of mice infected with Pneumocystis carinii were examined on both lung homogenates and bronchoalveolar lavage (BAL) fluids. In the lungs of infected mice, the number of P. carinii cysts rapidly decreased by day 7, then started to increase with a peak on day 14, and thereafter decreased gradually. When the presence of P. carinii was examined at the DNA level by dot blot hybridization, a similar clearance curve was obtained, and the organisms were shown to be completely eliminated on day 28. In the late phase of infection, leukocytes, mainly lymphocytes, increased in number when analyzed on lung homogenates, while no significant increase of inflammatory cells was observed in BAL fluids. An accumulation of both CD4+ and CD8+ T cells and an increase of activated T cells expressing IL-2R alpha were observed in lung homogenates of the infected mice. In addition, a considerable amount of IFN-gamma was detected in lung homogenates, but not in BAL fluids. These data indicate that lung homogenates are more suitable than BAL fluids for the analysis of cellular and cytokine responses in the lungs of mice infected with P. carinii. To define the involvement of IFN-gamma in host defense against P. carinii, the effect of this cytokine on the killing activity of macrophages against P. carinii was examined in vitro. IFN-gamma was found to augment this activity by increasing nitric oxide synthesis of the macrophages. Thus, it is suggested that IFN-gamma plays an important role in the protection of mice from P. carinii infection.