AICA (5-amino-4-imidazolecarboxamide)-riboside is taken up by isolated rat hepatocytes and converted by adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 126.96.36.199) into AICAR (ZMP), an intermediate of the de novo synthesis of purine nucleotides. We investigated if, in these cells, a cycle analogous to the adenosine-AMP substrate cycle operates between AICAriboside and ZMP. When 50 microM ITu, an inhibitor of adenosine kinase, was added to hepatocytes that had metabolized AICAriboside for 30 min, the concentration of ZMP decreased immediately. This was mirrored by a reincrease of AICAriboside. Rates of the ITu-induced decrease of ZMP and the increase of AICAriboside, calculated at different concentrations of ZMP, were first order, up to the highest concentration of ZMP (approx. 5 mumol/g of cells). Dephosphorylation of ZMP added to crude cytosolic extracts of rat liver displayed hyperbolic kinetics, with a Vmax of 0.65 mumol/min per g protein and an apparent Km of 5 mM, and was markedly inhibited by Pi, an inhibitor of IMP-GMP 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 188.8.131.52). We conclude that hepatocyte ZMP is continuously dephosphorylated, most likely by IMP-GMP 5'-nucleotidase, into AICAriboside, which is rephosphorylated into ZMP by adenosine kinase. Substrate cycling was also shown to occur between other nucleoside analogs and their phosphorylated counterparts.