The location with respect to the plasma membrane of aspartate 821 in erythrocytic anion exchanger has been determined by labeling inside-out vesicles and intact erythrocytes with impermeant reagents and following the outcome by site-directed immunochemistry. Intact erythrocytes and inside-out vesicles in the same container were vectorially modified with 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide and [³⁵S]sulfanilic acid. The inside-out vesicles were separated from the erythrocytes by differential centrifugation, and both the vesicles and membranes made from the erythrocytes were stripped with alkali and digested with trypsin to liberate from each sample the peptide YHPDVPYVK containing aspartate 821. The tryptic digests were passed over an immunoadsorbent specific for peptides with the amino-and carboxy-terminal sequences YHPD− and −PYVK. Specifically bound peptides were eluted with acid, and the eluates were pooled and submitted to high-pressure liquid chromatography. A peak of absorbance at 229 nm corresponding to the peptide YHPDVPYVK was present in chromatograms of samples from both the inside-out vesicles and the intact erythrocytes. Another peak that displayed absorbance at 229 and 250 nm, corresponding to the peptide YHP(p-[³⁵S]sulfo-β-aspartanilide)VPYVK, was observed in the chromatogram of the sample from the inside-out vesicles but not in the chromatogram of the sample from the erythrocytes. This peak had associated with it a large number of counts per minute of [³⁵S]sulfur, whereas no counts per minute of [³⁵S]sulfur above background were detected on the chromatogram of the sample from the erythrocytes. The incorporation of [³⁵S]sulfanilic acid into aspartate 821 of anion exchanger in inside-out vesicles was at least 10-fold greater than the incorporation of [³⁵S]sulfanilic acid into aspartate 821 of anion exchanger in erythrocytes when the two preparations were labeled in the same solution. These results demonstrate that aspartate 821, found between two hydrophobic segments in the sequence of anion exchanger, is located on the cytoplasmic surface of this membrane-spanning protein.