The affinities of a large number of deoxy and fluorodeoxy sugars for the glucose binding site in glycogen phosphorylase have been measured, and polarities and relative strengths of the hydrogen bonds at each position have been predicted on the basis of these data. Comparison with the recently refined X-ray crystal structure of the phosphorylase-glucose complex shows a generally good correlation between predicted and observed bond strengths, vindicating this approach to the evaluation of hydrogen bonding. Estimates of the net contributions of hydrogen bonds of different types (neutral-neutral and neutral-charged) are essentially identical with those obtained by a complementary approach on the tyrosyl tRNA synthetase-substrate complex [Fersht, A. R., Shi, J. P., Knill-Jones, J., Lowe, D. M., Wilkinson, A. J., Blow, D. M., Brick, P., Cortes, P., Waye, M. M. Y., & Winter, G. (1985) Nature (London) 314, 235-238]. The carbohydrate binding site structure determined is compared with that recently determined for the arabinose binding protein.