The antidiabetic activities of vanadium(V) and -(IV) prodrugs are determined by their ability to release active species upon interactions with components of biological media. The first X-ray absorption spectroscopic study of the reactivity of typical vanadium (V) antidiabetics, vanadate ([V<
sup>
V<
/sup>
O<
sub>
4<
/sub>
]<
sup>
3?<
/sup>
, A) and a vanadium(IV) bis(maltolato) complex (B), with mammalian cell cultures has been performed using HepG2 (human hepatoma), A549 (human lung carcinoma), and 3T3-L1 (mouse adipocytes and preadipocytes) cell lines, as well as the corresponding cell culture media. X-ray absorption near-edge structure data were analyzed using empirical correlations with a library of model vanadium(V), -(IV), and -(III) complexes. Both A and B ([V] = 1.0 mM) gradually converged into similar mixtures of predominantly five- and six-coordinate VV species (~75% total V) in a cell culture medium within 24 h at 310 K. Speciation of V in intact HepG2 cells also changed with the incubation time (from ~20% to ~70% V<
sup>
IV<
/sup>
of total V), but it was largely independent of the prodrug used (A or B) or of the predominant V oxidation state in the medium. Subcellular fractionation of A549 cells suggested that V<
sup>
V<
/sup>
reduction to V<
sup>
IV<
/sup>
occurred predominantly in the cytoplasm, while accumulation of V<
sup>
V<
/sup>
in the nucleus was likely to have been facilitated by noncovalent bonding to histone proteins. The nuclear V<
sup>
V<
/sup>
is likely to modulate the transcription process and to be ultimately related to cell death at high concentrations of V, which may be important in anticancer activities. Mature 3T3-L1 adipocytes (unlike for preadipocytes) showed a higher propensity to form V<
sup>
IV<
/sup>
species, despite the prevalence of V<
sup>
V<
/sup>
in the medium. Lastly, the distinct V biochemistry in these cells is consistent with their crucial role in insulin-dependent glucose and fat metabolism and may also point to an endogenous role of V in adipocytes.