Abstract Previous physical studies from this laboratory by sedimentation velocity and diffusion and by sedimentation equilibrium (Yue, R. H., Ratliff, R. L., and Kuby, S. A. (1967) Biochemistry 6, 2923–2932) have permitted an assignment of the kinetic molecular weight of the crystalline brewers' yeast nucleoside diphosphokinase, namely 102,000 (±2,000) at infinite dilution. The observed concentration dependence of its weight average molecular weight value (Yue et al.) was likely, in part, the result of a complex set of association-dissociation equilibria. Evidence presented here and derived (a) from the amino acid analyses, tryptic peptides, and terminal group analyses; (b) from sedimentation equilibrium studies on the native enzyme and on its S-carboxymethylated derivative, in the presence of disruptive agents; (c) from polyacrylamide gel electrophoresis in 0.2% dodecyl sulfate; and (d) from gel filtration through 8% agarose in 6 m guanidinium chloride, all indicate that the protein molecule is composed of six similar, if not identical, subunits. The best value for the individual subunit molecular weight (of this hexameric structure) may be selected from its amino acid composition, 17,300 ± 500. The NTP-NDP transphosphorylase appears to be unique among the other ATP-transphosphorylases studied in this laboratory, in that it possesses a single, shielded sulfhydryl group per subunit polypeptide chain, and in that it displays unusual resistance to inactivation by guanidinium chloride.