The molecular weight of adrenal iron-sulfur protein (adrenodoxin) is approximately 12,000 as determined by sedimentation-diffusion, by sedimentation-equilibrium, and from amino acid composition of the protein. The partial specific volume is 0.70 ml/g at 20°, and the diffusion coefficient, D20,w, is 11.2 × 10-7 cm2 sec-1. As described previously, the sedimentation coefficient, s20,w, is 1.55 S. The frictional ratio, f/f0, was calculated to be 1.29. The intrinsic viscosity is 3.0 ml/g at 22°. Therefore, the protein is a globular protein with the value of a/b (for major to minor axis of an ellipsoid) = about 5. The adrenal iron-sulfur protein contains approximately 100 amino acid residues with an abundance of acidic amino acids and a paucity of aromatic amino acids. From the ultraviolet optical rotatory dispersion, circular dichroism, and infrared absorption measurements, a gross difference in protein conformation between adrenal and spinach iron-sulfur proteins was found. © 1969, American Chemical Society. All rights reserved.