A recently discovered vesicular transport factor, termed p115, is required along with N-ethylmaleimidesensitive fusion protein (NSF) and soluble NSF attachment proteins for in vitro Golgi transport. p115 is a peripheral membrane protein found predominantly on the Golgi. Biochemical and electron microscopic analyses indicate that p115 is an elongated homodimer with two globular "heads" and an extended "tail" reminiscent of myosin II. We have cloned and sequenced cDNAs for bovine and rat p115. The predicted translation products are 90%o identical, and each can be divided into three domains. The predicted 108-kDa bovine protein consists of an N-terminal 73-kDa globular domain followed by a 29-kDa coiled-coil dimerization domain, a linker segment of 4 kDa, and a highly acidic domain of 3 kDa. p115 is related to Usolp, a protein required for endoplasmic reticulum to Golgi vesicular transport in Saccharomyces cerevisiae, which has a similar "headcoil-acid" domain structure. The p115 and Usolp heads are similar in size, have -25% sequence identity, and possess two highly homologous regions (62% and 60%o identity over 34 and 53 residues, respectively). There is a third region of homology (50%o identity over 28 residues) between the coiled-coil and acidic domains. Although the acidic nature of the p115 and Usolp C termini is conserved, the primary sequence is not. We discuss these results in light of the proposed function of p115 in membrane targeting and/or fusion.