After finding that rat liver mitochondria reduce arsenate (AsV) to the more toxic arsenite (AsIII), it was of interest to know if other cell fractions also carried out this process. Postmitochondrial supernatant (PMSN), isolated from rat liver, reduced AsV to AsIII only in the presence of a thiol. Dithiothreitol (DTT) supported the reduction much more effectively than glutathione. Separation of PMSN into microsome and cytosol revealed that the AsV reducing activity resided in the cytosol. AsV-like oxyanions, e.g., phosphate (P(i)) and vanadate, as well as mercurial thiol reagents inhibited the cytosolic AsV reducing activity, indicating the involvement of a P(i)-utilizing SH enzyme. On searching for a reduction partner, it was found unexpectedly that oxidized pyridine nucleotides (NAD(+) or NADP(+)), but not their reduced forms, increased AsIII formation. Some other purine nucleotide derivatives (e.g., AMP, GMP), but not pyrimidine nucleotides, also increased the rate 2-3-fold. Examination of the effect of nucleosides and nucleobases on AsV reduction yielded dramatic results: purine nucleosides (inosine or guanosine) increased the reduction 80-100-fold, whereas purine bases (hypoxanthine or guanine) decreased it 80-90%. Although the retentate obtained by ultrafiltration of cytosol was almost inactive, its AsV reductase activity could be regained by adding the filtrate or inosine or guanosine to the retentate, indicating that endogenous purine nucleosides were essential for AsV reduction by the cytosol. The hepatic cytosol of mice, hamsters, guinea pigs, and rabbits also exhibited AsV reductase activities in the presence of DTT, which were dramatically enhanced by inosine. Thus, the hepatic cytosol of all tested species can reduce AsV to AsIII. The reduction requires the presence of an appropriate thiol as well as a purine nucleoside (inosine or guanosine) and is inhibited by thiol reagents, the AsV analogue phosphate, and purine bases. Characterization of this AsV reductase activity led us to identification of a cytosolic AsV reductase, which is presented in the accompanying paper.