To determine the role of surfactant protein-A (SP-A) in host defense, the murine SP-A locus was targeted by homologous recombination to produce mice lacking SP-A. SP-A^−/− and wild-type mice were infected with mucoid Pseudomonas aeruginosa by intratracheal instillation. Pulmonary bacterial loads were greater in SP-A^−/− than in wild-type mice, with increased numbers of mucoid P. aeruginosa in lung homogenates at 6 and 24 h after infection. Pulmonary infiltration with polymorphonuclear leukocytes (PMN) was similar in both groups; however, an earlier influx of PMN into the lung occurred in the SP-A^−/− mice. The number of bacteria phagocytosed by alveolar macrophages was decreased in the SP-A^−/− mice at 1 h after infection. Superoxide-radical generation by PMN was similar for the SP-A^−/− and wild-type mice, but nitrite levels were increased in SP-A^−/− mice. Concentrations of tumor necrosis factor-α, interleukin-6, and macrophage inflammatory protein-2 (proinflammatory cytokines) were greater in bronchoalveolar lavage fluid at 2 h after infection in SP-A^−/− mice. SP-A plays an important role in the pathogenesis of mucoid P. aeruginosa infection in the lung in vivo by enhancing macrophage phagocytosis and clearance of bacteria, and by modifying the inflammatory response.