Clathrin and the -adaptin subunit of the AP-1 clathrin adaptor have been previously identified on H-K-ATPase-rich tubulovesicles from gastric acid secretory (oxyntic) cells [C. T. Okamoto, S. M. Karam, Y. Y. Jeng, J. G. Forte, and J. Goldenring. Am. J. Physiol. 274 (Cell Physiol. 43): C1017-C1029]. We further characterized this AP-1 adaptor from rabbit and hog tubulovesicles biochemically and immunologically. Clathrin coat proteins were stripped from purified tubulovesicular membranes and fractionated by hydroxyapatite chromatography. The AP-1 adaptor appears to elute at 200 mM sodium phosphate, based on the presence of proteins in this fraction that are immunoreactive with antibodies against three of the four subunits of this heterotetrameric complex: the -, µ₁-, and ₁-adaptin subunits. Although the putative -adaptin subunit in this fraction is not immunoreactive with the anti--adaptin monoclonal antibody (MAb), this -adaptin is immunoreactive with polyclonal antibodies (PAbs) directed against the peptide sequence Gly⁶²⁵-Asp-Leu-Leu-Gly-Asp-Leu-Leu-Asn-Leu-Asp-Leu-Gly-Pro-Pro-Val⁶⁴⁰, a region conserved between ₁- and ₂-adaptins that is thought to be involved in the binding of clathrin heavy chain. Immunoprecipitation of the AP-1 adaptor complex from this fraction with anti--adaptin MAb 100/3 resulted in the coimmunoprecipitation of the -adaptin that did not react with the anti--adaptin MAb but did react with the anti--adaptin PAbs. In contrast, immunoprecipitation of the AP-1 adaptor complex from crude clathrin-coated vesicles from brain resulted in the coimmunoprecipitation of a -adaptin that was recognized by both the anti--adaptin MAb and PAbs. These results suggest that the tubulovesicular AP-1 adaptor complex may be distinct from that found in the trans-Golgi network and may contain an immunologically distinct -adaptin. This immunologically distinct -adaptin may be diagnostic of apical tubulovesicular endosomes of epithelial cells.