Rab3D is a small GTP-binding protein associated with secretory vesicles in various exocrine and endocrine cells, where it has been implicated in regulated exocytosis. Data obtained previously in pancreas have suggested that rab3D is involved in the coating of secretory granules with filamentous actin. In the present study we employed Western blot analysis, immunofluorescence, and immunoelectron microscopy to examine the distribution of rab3D in rat lung. Rab3D immunoreactivity was detected in bronchiolar Clara cells and alveolar epithelial type II (AET-II) cells. In both cell types, rab3D displayed preferential localization to secretory vesicles that were identified using specific antibodies against Clara Cell Secretory Protein and p180 lamellar body protein, respectively. Interestingly, rab3D was associated with only 24% of the lamellar bodies in AET-II cells. Rab3D-positive lamellar bodies were typically in close proximity of the apical plasma membrane, where exocytosis occurs. Another subpopulation of lamellar bodies, constituting only 2%, was not only rab3D-positive but could also be labeled with the filamentous-actin probe phalloidin. A third subpopulation, constituting 9%, displayed actin coating without rab3D staining. We propose that these three lamellar body subpopulations represent consecutive intermediates along the regulated exocytotic pathway, implying that rab3D release and actin coating are intimately linked processes.