Cyclic guanidino-sugars with different pK(a) values are designed and synthesized as transition-state analog inhibitors of galactosidases. Characterization of these structures (7, 10, 12) reveals that 7 and 10 are in a pH-dependent equilibrium between a furanose form and a mixture of neutral and protonated tetrahydropyrimidine forms. In contrast, the O-linked guanidino-sugar 12 exists as the tetrahydropyrimidine forms above pH 5. The furanose-tetrahydropyrimidine equilibrium can thus be modulated with the appropriate N-substituent which affects the guanidino-sugar pK(a) value. Enzymatic inhibition by 7, 10, and 12 is also pH-dependent, indicating that the enzymes recognize the tetrahydropyrimidine form. Evidence is presented to support a dominant role for the uncharged form of the six-membered cyclic guanidino-sugar in the inhibition of galactosidases. Though the inhibition potency is moderate (K-i range 4-50 mu M), the use of cyclic guanidino-sugars in the study provides new insights into the mechanism of inhibition of glycosidases.