Rab3D is a small GTP-binding protein that associates with secretory granules of endocrine and exocrine cells. The physiological role of Rab3D remains unclear. While it has initially been implicated in the control of regulated exocytosis, recent deletion-mutation studies have suggested that Rab3D is involved in the biogenesis of secretory granules. We here report the unexpected finding that Rab3D also associates with early Golgi compartments in intestinal goblet cells and in Brunner's gland acinar cells. Expression of Rab3D in intestine was demonstrated by SDS-PAGE and Western blotting of homogenates prepared from rat duodenum and colon. Confocal laser-scanning microscopy revealed Rab3D immunofluorescence in the Golgi area of goblet cells of duodenum and colon, and in Brunner's gland acinar cells. There was no co-localization between Rab3D and the trans-Golgi Network marker, TGN-38. In contrast, Rab3D co-localized partially with the cis-Golgi marker, GM-130, and with the marker of cis-Golgi and COPI vesicles, -COP. Strong co-localization was observed between Rab3D and the lectins, Griffonia simplicifolia agglutinin II and soybean agglutinin, which have been described as markers of medial- and cis-Golgi, respectively. Rabphilin, a putative effector of Rab3D, displayed an identical pattern of Golgi localization. Incubation of colon tissue with carbamylcholine or deoxycholate to stimulate exocytosis by goblet cells caused a partial redistribution of Rab3D to the cytoplasm and mucous granule field, and a concomitant transformation of the Golgi architecture. Taken together, the present data suggest that Rab3D and Rabphilin may regulate the secretory pathway at a much earlier stage than what has hitherto been assumed.