Using small-angle X-ray scattering from solutions of yeast hexokinase, we have measured the radii of gyration of the monomeric B isozyme and its complexes with sugar substrates. We find that the radius of gyration decreases by 0.95 +/- 0.24 A upon binding glucose and 1.25 +/- 0.28 A upon binding glucose 6-phosphate. This observed reduction in radius of gyration in the presence of glucose is the same as that calculated from the coordinates of the high-resolution crystal structures of native hexokinase B and a glucose complex with hexokinase A. Thus, these measurements suggest that the dramatic closing of the slit between the two lobes of hexokinase observed in the crystal structures (Bennett, W.S., & Steitz, T.A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848--4852) occurs in solution when either glucose or glucose 6-phosphate is bound.