The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the differentiation of dendritic cells (DCs) during pulmonary viral infection was investigated by using a mouse model of GM-CSF transgene expression established with an adenoviral vector (AdGM-CSF). GM-CSF gene transfer resulted in increased levels of GM-CSF in the lung, which peaked at day 4 and remained increased up to day 19. A striking cellular response composed predominantly of macrophage-like cells was observed in the lung receiving AdGM-CSF but not control vector. By FACS analysis, the majority of these cells were identified at an early time point as macrophages and later as mature/activated myeloid DCs characterized by CD11bbright, CD11cbright, MHC class IIbright, and B7.1bright. In contrast, GM-CSF had a weak effect on a small DC population that was found present in normal lung and was characterized by CD11cbright and CD11blow. By immunohistochemistry staining for MHC II, the majority of activated antigen-presenting cells were localized to the airway epithelium and peribronchial/perivascular areas in the lung. A concurrently enhanced Th1 immune response was observed under these conditions. The number of CD4 and CD8 T cells was markedly increased in the lung expressing GM-CSF, accompanied by increased release of interferon (IFN)γ in the lung. Furthermore, lymphocytes isolated from either lung parenchyma or local draining lymph nodes of these mice but not the control mice released large amounts of IFNγ on adenoviral antigen stimulation in vitro. These findings reveal that GM-CSF promotes the differentiation and activation of a myeloid DC population primarily by acting on macrophages during pulmonary immune responses.