Here, we report the localization and characterization of BHKp23, a member of the p24 family of transmembrane proteins, in mammalian cells. We find that p23 is a major component of tubulovesicular membranes at the cis side of the Golgi complex (estimated density: 12,500 copies/μm² membrane surface area, or ≈30% of the total protein). Our data indicate that BHKp23-containing membranes are part of the cis-Golgi network/intermediate compartment. Using the G protein of vesicular stomatitis virus as a transmembrane cargo molecule, we find that p23 membranes are an obligatory station in forward biosynthetic membrane transport, but that p23 itself is absent from transport vesicles that carry the G protein to and beyond the Golgi complex. Our data show that p23 is not present to any significant extent in coat protein (COP) I-coated vesicles generated in vitro and does not colocalize with COP I buds and vesicles. Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment. Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis- Golgi network/intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules. These observations together with the fact that p23 is a highly abundant component in the intermediate compartment, lead us to propose that p23 contributes to membrane structure, and this contribution is necessary for efficient segregation and transport.