Although proinflammatory cell signaling in the alveolo-capillary region predisposes to acute lung injury, key cell-signaling mechanisms remain inadequately understood. Alveolo-capillary inflammation is likely to involve coordinated signaling among cells of different phenotypes. For example, migration of inflammatory cells into the alveolus might entail coordinated signaling between adjoining alveolar epithelial and microvascular endothelial cells. The popular cultured cell experimental strategy fails to replicate this multicellular environment. Cultured lung cells, both alveolar and endothelial, undergo phenotypic transformations; hence they might inadequately reflect innate responses of native cells. Consequently, new approaches are required for the investigation of cell signaling in the native setting. Here we summarize new developments in classical intravital microscopy and discuss real-time fluorescence imaging as a novel technique for studying second-messenger mechanisms in the alveolo-capillary region.