Cyclic adenosine monophosphate (cAMP) was found to decrease the availability of 5-phosphoribosyl-1-pyrophosphate (PRPP) and to decelerate the rate of de novo purine synthesis in suspensions of adult rat hepatocytes. Glucagon did not affect these parameters. The glucagon antagonist des-His1[Glu9]glucagon amide (DHGA), and the protein kinase C activator 1,2-dioctanoyl-sn-glycerol (DOG) were also found to lower PRPP availability. Incubation of the hepatocytes with dbcAMP or with DHGA, did not alter the activity of PRPP synthetase in the hepatocyte lysates, indicating that the above effects are not mediated through the activity of this enzyme. The possibility that the decrease in PRPP availability reflects increased consumption associated with accelerated pyrimidine synthesis is discussed. The decelerated rate of de novo purine synthesis is probably secondary to the decreased PRPP availability.