A joint X-ray crystallographic (1.3 Angstrom resolution) and ab initio quantum mechanical analysis of a uridine vanadate-ribonuclease A complex (W-RNase A) is undertaken to probe specific aspects of the microscopic mechanism by which ribonuclease functions to catalyze the hydrolysis of its natural substrate, phosphate esters. Comparison of the structural features of the vanadate portion from the final X-ray refinement with the oxy-vanadate model compounds determined computationally provides direct evidence of the likely protonation state of the UV inhibitor bound in the active site. Specifically, the UV bound in the active site of UV-RNase A is found to be monoanionic, and the most likely source of this proton is from the active site residue His12. Together with the structural data, these results strongly suggest that even though His12 may act as the catalytic base in the first step of ale mechanism, transphosphorylation, and the catalytic acid in the second step, hydrolysis, it must also play an additional, although perhaps secondary, role in stabilizing the pentacoordinate phosphorane structure through proton transfer. On the basis of its close proximity to critical vanadate oxygen in the UV, and data obtained from a previous computational study, Lys41 is Likely to play a more intimate role in the catalytic mechanism than previously proposed, potentially acting as the catalytic base in certain cases. Two possible detailed microscopic mechanisms are presented.