Reduction of arsenate (AsV) to the more toxic arsenite (AsIII) is of high toxicological importance, yet in vivo relevant enzymes involved have not been identified. Purine nucleoside phosphorylase (PNP) is an efficient AsV reductase in vitro, but its role in AsV reduction is irrelevant in vivo. Intact human red blood cells (RBC) possess an AsV reductase activity that is PNP-independent, diminished by depletion of glutathione (GSH), enhanced by oxidants of erythrocytic NAD(P)H, and possibly linked to the lower part of the glycolytic pathway. In order to characterize this PNP-independent AsV reductase activity further, we examined the effects of GSH, inorganic phosphate, some inhibitors of glucose metabolism, glycolytic substrates, and pyridine, as well as adenine nucleotides on AsV reduction in lysed RBC and rat liver cytosol in the presence of BCX-1777, a PNP inhibitor. In hemolysate, GSH enhanced AsV reduction in a concentration-dependent manner, whereas phosphate inhibited it. Glycolytic substrates, especially fructose-1,6-bisphosphate and phosphoglyceric acids, improved AsV reductase activity. NAD, especially together with these substrates, strongly increased AsIII formation, whereas NADH strongly inhibited it. NADP and adenine nucleotides diminished, while 2-phosphoglycollate, which increases the breakdown of the RBC-specific compound 2,3-bisphosphoglycerate to 3-phosphoglycerate, doubled the AsV reductase activity. Although AsV reduction by the liver cytosol responded similarly to GSH, NAD, and glycolytic substrates as in the hemolysate, it was barely influenced by NADH, was diminished by 2-phosphoglycollate, and was stimulated by NADP. Collectively, hemolysate and rat liver cytosol possess a PNP-independent AsV reductase activity. This enzymatic activity requires GSH, NAD, and glycolytic substrates, and purportedly involves one or both of the two functionally linked glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. In addition, the data presented here suggest that yet another PNP-independent AsV reductase resides in the hepatic cytosol. Although this latter enzyme remains unknown, identification of the AsV reductase depending on GSH, NAD, and glycolytic substrates is presented in the following paper.