The orientation of the nitroxide moiety of an isotopically substituted spin-labeled derivative of dihydrostilbenedisulfonate ([15N,2H13]-SL-H2DADS-maleimide) covalently coupled at the extracellular stilbenedisulfonate binding site of the human erythrocyte anion exchange protein, band 3, has been determined relative to the membrane normal axis of intact cells. The X-band linear electron paramagnetic resonance (EPR) spectra of [15N,2H13]-SL-H2DADS-maleimide-labeled band 3 in intact erythrocytes oriented by flow through an EPR flat cell have been obtained for two orthogonal orientations of the sample in the DC magnetic field. Two different methods of analysis have provided very similar values for the angles alpha 1 and beta 1 which uniquely define the orientation of the nitroxide axis frame relative to the membrane normal axis. In the first approach, a variable fraction of the cells, f, were taken to be biconcave disks perfectly oriented relative to the flat cell surface with the remainder, 1-f, isotropically oriented. Simultaneous nonlinear least squares analysis of the spectra obtained at the two sample orientations yielded best fit values of f = 0.60, alpha 1 = 58 degrees, and beta 1 = 36 degrees. In the second approach, the EPR spectra of flow-oriented intact erythrocytes labeled with the fatty acid spin-label, [15N,2H12]-5-nitroxyl stearate, have been obtained at the two sample orientations. These two spectra have been used to determine a model-independent distribution of membrane normal orientations in the sample. Using this experimentally determined membrane normal orientation distribution, the EPR spectra of [15N,2H13]-SL-H2DADS-maleimide-labeled erythrocytes were then reanalyzed to obtain a second determination of the nitroxide orientation, alpha 1 = 61 degrees and beta 1 = 37 degrees. The orientation of the nitroxide with respect to the membrane normal axis determined in the present study is nearly identical to the orientation of the nitroxide with respect to the uniaxial rotational diffusion axis, alpha = 66 degrees and beta = 34 degrees, as determined from saturation transfer EPR (ST-EPR) studies [Hustedt, E. H., & Beth, A. H. (1995) Biophys. J. 69, 1409-1423]. This result supports the conclusion that the motion observed using ST-EPR spectroscopy is, in fact, the uniaxial rotational diffusion of band 3 about the membrane normal.