Band 3, the predominant membrane-spanning polypeptide and purported anion transport protein of human red cells, was isolated by a new procedure which utilized selective solubilization and anion exchange chromatography on Affi-Gel 102 in 0.5% and Triton X-100/0.03% sodium dodecyl sulfate. Rabbit anti-serum prepared against the purified protein reacted with human and monkey band 3 but gave no immunoprecipitate with membrane proteins from several non-primate species. The antiserum was directed solely towards a portion of the cytoplasmic pole of the band 3 polypeptide contained within a 23,000 dalton amino-terminal fragment, as shown by agglutination, absorption, double diffusion and immunoprecipitation techniques. Saturation of both surfaces of resealed erythrocyte ghosts with the anti-band 3 antiserum had no significant effect on chloride transport. Our data define the topographically-limited immunogenicity of human band 3 in rabbits, demonstrate a lack of immunological cross-reactivity of band 3 between primates and non-primates, and support the hypothesis that the cytoplasmic domain of band 3 is not intimately involved in anion transport.