Transport of proteins between intracellular membrane compartments is a highly regulated process that depends on several cytosolic factors. By using the well characterized intra-Golgi cell-free transport assay, we purified from bovine brain cytosol a 56-kDa protein that shows a significant transport activity. Partial sequencing of four tryptic peptides obtained from the 56-kDa protein revealed its identity to a cytosolic protein previously characterized as a selenium-binding protein, SBP56. Recombinant SBP56 expressed in Escherichia coli exhibited transport activity when added to the cell-free intra-Golgi transport. Affinity purified anti-SBP56 polyclonal antibodies specifically inhibited intra-Golgi transport in vitro. Although SBP56 is predominantly localized in the cytosol, a significant amount is associated with membranes. Subcellular fractionation showed that this protein is peripherally associated with the Golgi membrane. The experiments presented in this study indicate that SBP56 participates in late stages of intra-Golgi protein transport.