Adenovirus is endocytosed and efficiently destroyed by human and murine alveolar macrophages (AMs) and rapidly cleared from the lungs of wild-type but not GM-CSF(-/-) mice. We hypothesized that GM-CSF may regulate adenovirus clearance in AMs via the transcription factor PU.1 by redirecting virion trafficking from the nucleus to lysosomes. This hypothesis was tested in murine AM cell lines with altered GM-CSF and/or PU.1 expression including MH-S (GM-CSF(+/+)PU.1(Pos)), mAM (GM-CSF(-/-)/PU.1(Neg)), and mAM(PU.1+) (GM-CSF(-/-)/PU.1(Pos); PU.1-transduced mAM cells) and A549 (an epithelial-like cell line) using a human adenovirus expressing a beta-galactosidase reporter. In PU.1(Neg) mAM and A549 cells, adenovirus efficiently escaped from endosomes, translocated to the nucleus, and expressed the viral reporter in most cells. In marked contrast, in PU.1(Pos) mAM(PU.1+) and MH-S cells, adenovirus failed to escape from endosomes, colocalized exclusively with endosome/lysosome markers (Rab5, Rab7, and Lamp1), and rarely expressed the reporter. Retroviral expression of PU.1 in A549 cells blocked endosomal escape, nuclear translocation and reporter expression. Inhibition of endosome acidification also blocked escape, nuclear translocation, and reporter expression in PU.1(Neg) cells. The effect of PU.1 on viral trafficking and transduction could not be explained by an effect on endosome acidification or on differences in viral load. PU.1 reduced expression of integrin beta(5), a host factor important for endosomal escape of adenovirus, suggesting that PU.1 redirects adenoviral trafficking by modulating integrin signaling. These results demonstrate that PU.1 uncouples infection from internalization in AMs, providing a mechanism for AMs to avoid infection by adenovirus during clearance.