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Electron paramagnetic resonance (EPR) and saturation transfer–EPR (ST–EPR) techniques were employed to investigate the hydrodynamic properties of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Both apo- and holoenzyme were spin-labeled at the active site cysteine-149 residue with N- (1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) - maleimide. The apo- and holoenzymes were observed to have the same hydrodynamic structure and the spectroscopic results were consistent with these complexes behaving as spheres with hydrated radii of 41 Å. The environment of the paramagnetic electron was significantly more polar in the spin-labeled holoenzyme than in the spin-labeled apoenzyme, suggesting that either ionic residues are positioned closer to the active site in the holoenzyme or that ionic segments of coenzyme nicotinamide adenine dinucleotide (NAD+) itself may interact with the paramagnetic electron of the maleimide spin label. The dependence of the phase quadrature second harmonic absorption ST-EPR signal upon microwave and Zeeman modulation field intensities was examined with particular attention directed to effects of the variation of these fields over samples of finite dimensions. Such considerations are crucial when comparing data obtained employing different microwave resonators or different sample geometries within the same resonator. The problem of accurately establishing phase nulls for phase quadrature measurements was examined as was the problem of estimating phase resolution. Thus, the present work represents both a specific application and a generalization of the ST-EPR methodology proposed by Thomas, Dalton, and Hyde [J. Chem. Phys. 65, 3006 (1976)].