Recent studies have described a widely expressed adaptor-like complex, named AP-3, which is likely involved in protein sorting in exocytic/endocytic pathways. The AP-3 complex is composed of four distinct subunits. Here, we report the identification of one of the subunits of this complex, which we call beta3A-adaptin. The predicted amino acid sequence of beta3A-adaptin reveals that the protein is closely related to the neuron-specific protein beta-NAP (61% overall identity) and more distantly related to the beta1- and beta2-adaptin subunits of the clathrin-associated adaptor complexes AP-1 and AP-2, respectively. Sequence comparisons also suggest that beta3A-adaptin has a domain organization similar to beta-NAP and to beta1- and beta2-adaptins. beta3A-adaptin is expressed in all tissues and cells examined. Co-purification and co-precipitation analyses demonstrate that beta3A-adaptin corresponds to the approximately 140-kDa subunit of the ubiquitous AP-3 complex, the other subunits being delta-adaptin, p47A (now called mu3A) and sigma3 (A or B). beta3A-adaptin is phosphorylated on serine residues in vivo while the other subunits of the complex are not detectably phosphorylated. beta3A-adaptin is not present in significant amounts in clathrin-coated vesicles. The characteristics of beta3A-adaptin reported here lend support to the idea that AP-3 is a structural and functional homolog of the clathrin-associated adaptors AP-1 and AP-2.