Constitutive secretory vesicles carrying heparan sulfate proteoglycan (HSPG) were identified in isolated rat hepatocytes by pulse-chase experiments with [35S]sulfate and purified by velocity-controlled sucrose gradient centrifugation followed by equilibrium density centrifugation in Nycodenz. Using this procedure, the vesicles were separated from plasma membranes, Golgi, trans-Golgi network (TGN), ER, endosomes, lysosomes, transcytotic vesicles, and mitochondria. The diameter of these vesicles was approximately 100-200 nm as determined by electron microscopy. A typical coat structure as described for intra-Golgi transport vesicles or clathrin-coated vesicles could not be seen, and the vesicles were not associated with the coat protein beta-COP. Furthermore, the vesicles appear to represent a low density compartment (1.05-1.06 g/ml). Other constitutively secreted proteins (rat serum albumin, apolipoprotein E, and fibrinogen) could not be detected in purified HSPG-carrying vesicles, but banded in the denser fractions of the Nycodenz gradient. Moreover, during pulse-chase labeling with [35S]methionine, labeled albumin did not appear in the post-TGN vesicle fraction carrying HSPGs. These findings indicate sorting of HSPGs and albumin into different types of constitutive secretory vesicles in hepatocytes. Two proteins were found to be tightly associated with the membranes of the HSPG carrying vesicles: a member of the ADP ribosylation factor family of small guanine nucleotide-binding proteins and an unknown 14-kD peripheral membrane protein (VAPP14). Concerning the secretory pathway, we conclude from these results that ADP ribosylation factor proteins are not only involved in vesicular transport from the ER via the Golgi to the TGN, but also in vesicular transport from the TGN to the plasma membrane.