Prelabeled adenine nucleotides were rapidly degraded in freshly isolated rat liver cells incubated with glycerol at concentrations greater than 0.25 mM. Concomitantly, the [14C]ATP/[14C]ADP and [14C]ATP/[14C]AMP ratios were decreased. There was a transient increase in radioactive IMP, inosine, and hypoxanthine and a constant accumulation of allantoin. Radioactive adenosine also accumulated transiently under certain conditions. The intracellular Pi; concentration was decreased. The magnitude of most of these changes was dependent upon the concentration of glycerol. The effects of glycerol were similar to those of fructose. The extent of endogenous adenine nucleotide catabolism was smaller at high Pi concentration, whereas the extent of glycerol-induced catabolism was unaffected. However, less inosine and more hypoxanthine accumulated during glycerol-induced catabolism at high Pi concentration.