The autophosphorylation of interferon (IFN)-induced double-stranded RNA-dependent p68 protein kinase (PKR) and phosphorylation of the alpha-subunit of the translation initiation factor eIF-2 were inhibited by 10 mM 2-aminopurine in vitro. High concentrations of ATP overcame the inhibition. Kinetic studies indicated that 2-aminopurine is a competitive inhibitor with respect to ATP, suggesting that these two molecules bind the same site on the kinase. Treatment of HeLa cells with poly(I):poly(C) stimulated PKR autophosphorylation in vivo. The stimulated activity was inhibited by 10 mM 2-aminopurine to approximately the same extent as the in vitro inhibition.