The yeast coat protein II (COPII) is responsible for vesicle budding from the endoplasmic reticulum (ER). Mammalian functional homologues for all yeast COPII components, except for Sec31p, have been reported. We have cloned a mammalian cDNA whose product (Sec31A) is about 26% identical to Saccharomyces cerevisiae Sec31p. Data base searches also revealed another partial sequence encoding a polypeptide (Sec31B) that is 40% identical to Sec31A. Northern analysis revealed that Sec31A transcripts are ubiquitously and abundantly expressed, while Sec31B transcripts are particularly enriched in the testis and thymus, but present in very low levels in other tissues. Sec31A is localized to vesicular structures that scatter throughout the cell but are concentrated at the perinuclear region. The structures marked by Sec31A contain Sec13, a component of COPII that is well characterized to mark the ER exit sites. Immunoelectron microscopy revealed that Sec31A colocalizes with Sec13 in structures with extensive vesicular-tubular profiles. Antibodies raised against a C-terminal portion of Sec31A co-precipitate Sec13 and inhibit ER-Golgi transport of temperature-arrested vesicular stomatitis G protein in a semi-intact cell assay. Cytosol immunodepleted of Sec31A failed to support vesicular stomatitis G protein transport, which can be rescued by a high molecular weight fraction of the cytosol containing both Sec31A and Sec13. We conclude that Sec31A represents a functional mammalian homologue of yeast Sec31p.