The interactions of vanadate and its complexes of uridine, 5,6-dihydrouridine, and methyl β-D-ribofuranoside with bovine pancreatic ribonuclease A (RNase A) (EC 3.1.27.5) were studied by51V NMR spectroscopy and enzyme kinetics. From kinetic studies, it was found that neither inorganic vanadate nor the methyl β-D-ribofuranoside–vanadate complex significantly inhibited the RNase A catalyzed hydrolysis of uridine 2′,3′-cyclic monophosphate. The NMR binding studies were in full agreement with the kinetics studies and showed that neither inorganic vanadate nor the methyl β-D-ribofuranoside–vanadate complex was bound tightly by the enzyme. Approximate binding constants were (5.0 ± 1.0) × 10−7 M and (3.0 ± 0.6) × 10−6 M for the uridine–and 5,6-dihydrouridine–vanadate complexes, respectively. An induced-fit mechanism is suggested, in which the pyrimidine subsite of the active site of RNase A must be fully occupied for the enzyme to be able to tightly bind the transition state or transition state analog. Calculation of the binding energies of vanadate complexes in ribonuclease, phosphoglycerate mutase, and phosphoglucomutase revealed an excess of binding energy over the analogous phosphate derivative of about 25 kJ/mol for all enzymes, even though the binding constants themselves varied by about six orders of magnitude. This energy represents about 40% of that expected to be available for a trigonal-bipyramidal transition state and requires a reassessment of the role of vanadate as a transition state analogue for phosphate transfer.Keywords: vanadate, ribonuclease, transition state, binding constants, phosphate analogues, kinetics.