Although aryl hydroxamic acids are well-known to form coordination complexes with vanadate (V-V), the nature of these complexes at neutral pH and submillimolar concentrations, the conditions under which such complexes inhibit various serine amidohydrolases, is not well established. A series of qualitative and quantitative experiments, involving UV/vis, H-1 NMR, and V-51 NMR spectroscopies, established that both 1:1 and 1:2 vanadate/hydroxamate complexes form at pH 7.5, with the former dominating at submillimolar concentrations, Formation constants for the complexes of several aryl and alkyl hydroxamic acids were determined; for example, for benzohydroxamic acid, the stepwise formation constants of the 1:1 and 1:2 complexes were 3000 and 400 M-1, respectively, The V-51 chemical shift of the 1:1 4-nitrobenzohydroxamic acid complex was -497 ppm, and that of its unsubstituted analogue was -498 ppm. A H-1-(IN)-I-15 HSQC spectrum of the 4-nitrobenzo-N-15-hydroxamic acid/vanadate complex indicated the presence of an N-H group with N-15 and H-1 chemical shifts of 115 and 5.83 ppm, respectively. A C-13 NMR spectrum of the complex of 4-nitrobenzo-C-13-hydroxamic acid with vanadate displayed a resonance at 170.1 ppm and thus a coordination-induced shift (CIS) of +3.8 ppm. In contrast, the CIS value of an established 1:2 complex, thought to contain chelated hydroxamic acid ligands, was +11.9 ppm. These spectral data led to the following structural picture of 1:1 complexes of vanadate and aryl hydroxamic acids. They contain penta- or hexa-coordinated vanadium. The ligand is in the hydroxamate rather than hydroximate form. The ligand is presumably bound to vanadium through the hydroxamic hydroxyl oxygen, but the hydroxamic acid carbonyl oxygen interacts weakly with vanadium. These species are the most likely candidates for the inhibitors of serine amidohydrolases found in vanadate/ hydroxamic acid mixtures.