Activated alveolar macrophages (AMphi) are known to constitute a critical modulator of the lung inflammatory response through the production of various mediators. However, the role of activated AMphi in acute lung injury (ALI) and acute respiratory distress syndrome is less well known. To address this issue, we examined a lipopolysaccharide (LPS)-induced lung injury model for the role of activated AMphi in vivo, focusing on activation through CD40, which is one of the most important pathways for the activation of antigen-presenting cells. Without CD40, LPS-induced ALI was significantly reduced in its histological degree of injury and recruitment of neutrophils into the lung. In addition, the release in the lung of inflammatory mediators such as tumor necrosis factor-alpha, interleukin-1beta, macrophage inflammatory protein 2, or matrix metalloproteinase was significantly reduced in mice deficient in CD40 (CD40KO). To elucidate the mechanism of this attenuation of ALI in CD40KO mice, we studied the function of AMphi ex vivo. AMphi purified from CD40KO mice could not induce expression of inducible nitric oxide synthase (iNOS) by LPS, although iNOS in wild-type AMphi was induced by LPS independently of CD40-CD154 interaction. The loss of surface expression of CD40 was enough to interrupt the expression of iNOS in AMphi in response to LPS. Also based on the tissue nitrotyrosine staining, the reactive oxygen and nitrogen intermediates seemed to be reduced in tissue in CD40KO mice. These results indicated that activation of AMphi through CD40 might be involved not only in amplification by the interaction with CD154 but also in the development of ALI by CD40 itself, and that the functional blockade of CD40 would yield one of the targets for the treatment of LPS-induced ALI and acute respiratory distress syndrome.