A heterotrimeric Gai subunit, «i- 3 , is localized on Golgi membranes in LLC-PKI and NRK epithelial cells where it colocalizes with mannosidase II by immunofluorescence . The M-3 was found to be localized on the cytoplasmic face of Golgi cisternae and it was distributed across the whole Golgi stack. The a;_3 subunit is found on isolated rat liver Golgi membranes by Western blotting and Gai -3 on the Golgi apparatus is ADP ribosylated by pertussis toxin. LLCPKI cells were stably transfected with GO;-3 on an MT1, inducible promoter in order to overexpress ai-3 on Golgi membranes. The intracellular processing and constitutive secretion of the basement membrane heparan sulfate proteoglycan (HSPG) was measured in LLC-PK, cells. Overexpression of ai-3 on Golgi membranes in transfected cells retarded the secretion of HSPG and accumulated precursors in the medial-trans-Golgi . This effect was reversed by treatment of cells with pertussis toxin which results in ADP-ribosylation and functional uncoupling of Gai-3 on Golgi membranes. These results provide evidence for a novel role for the pertussis toxin sensitive Gai-3 protein in Golgi trafficking of a constitutively secreted protein in epithelial cells