To identify the species responsible for the insulin-mimetic activity of bis(acetylacetonato)oxovanadium(IV) [VO(acac)₂], we have investigated its interaction with bovine serum albumin (BSA) by EPR (electron paramagnetic resonance) and angle-selected ENDOR (electron nuclear double resonance), correlating results with assays of glucose uptake by 3T3-L1 adipocytes. In aqueous solutions of VO(acac)₂, two ionizations were detected with pK_a values of ~3.1 and ~1.7, governing formation of four spectral species (A - D) differing according to g- and A-values. Species A - C in aqueous solution and species A' - C' in methanol exhibited correspondingly identical g- and A-values, indicating similar species formed in both solvents. Species D detected only in aqueous solutions at very low pH was assigned to formation of [VO(H₂O)₅]²⁺. ENDOR spectra of A' and C' showed no change in inner-shell coordination structure at low and high [H⁺] in methanol. EPR spectra of VO(acac)₂ showed no broadening in the presence of BSA; however, ENDOR titrations of VO(acac)₂ in the presence of BSA were indicative of 1:1 VO(acac)₂ : albumin adduct formation. The influence of VO(acac)₂ on uptake of 2-deoxy-D-[1-¹⁴C]glucose by serum-starved 3T3-L1 adipocytes was measured in the presence and absence of BSA. Glucose uptake was stimulated 9-fold in the presence of 0.5 mM VO(acac)₂, compared to 17-fold in the presence of 0.5 mM VO(acac)₂ plus 1 mM BSA, and 22-fold in the presence of 100 nM insulin. BSA had no influence on glucose uptake, on the action of insulin, or on glucose uptake in the presence of VOSO₄. The maximum influence of VO(acac)₂ was observed at VO(acac)₂ : BSA ratios less than or equal to 1.0. Similar effects were observed also with bis(maltolato)oxovanadium(IV). These results suggest that the enhanced insulin-mimetic action of organic chelates of VO²⁺ is likely due to adduct formation with BSA and possibly other serum transport proteins.