Several lines of evidence suggest that glycophorin A (GPA) interacts with band 3 in human erythrocyte membranes including: i) the existence of an epitope shared between band 3 and GPA in the Wright b blood group antigen, ii) the fact that antibodies to GPA inhibit the diffusion of band 3, iii) the observation that expression of GPA facilitates trafficking of band 3 from the endoplasmic reticulum to the plasma membrane, and iv) the observation that GPA is diminished in band 3 null erythrocytes. Surprisingly, there is also evidence that GPA does not interact with band 3, including data showing that: i) band 3 diffusion increases upon erythrocyte deoxygenation whereas GPA diffusion does not, ii) band 3 diffusion is greatly restricted in erythrocytes containing the Southeast Asian Ovalocytosis mutation whereas GPA diffusion is not, and iii) most anti-GPA or anti-band 3 antibodies do not co-immunoprecipitate both proteins. To try to resolve these apparently conflicting observations, we have selectively labeled band 3 and GPA with fluorescent quantum dots in intact erythrocytes and followed their diffusion by single particle tracking. We report here that band 3 and GPA display somewhat similar macroscopic and microscopic diffusion coefficients in unmodified cells, however perturbations of band 3 diffusion do not cause perturbations of GPA diffusion. Taken together the collective data to date suggest that while weak interactions between GPA and band 3 undoubtedly exist, GPA and band 3 must have separate interactions in the membrane that control their lateral mobility.