Activation of the double-stranded RNA-dependent protein kinase (PKR) has been implicated in the pathogenesis of several neurodegenerative diseases. We find that a compound widely used as a pharmacological inhibitor of this enzyme, referred to as PKR inhibitor (PKRi), {8-(imidazol-4-ylmethylene)-6H-azolidino[5,4-g]benzothiazol-7-one}, protects against the death of cultured cerebellar granule and cortical neurons. PKRi also prevents striatal neurodegeneration and improves behavioral outcomes in a chemically induced mouse model of Huntington's disease. Surprisingly, PKRi fails to block the phosphorylation of eIF2alpha, a downstream target of PKR, and does not reduce the autophosphorylation of PKR enzyme immunoprecipitated from neurons. Furthermore, neurons lacking PKR are fully protected from apoptosis by PKRi, demonstrating that neuroprotection by this compound is not mediated by PKR inhibition. Using in vitro kinase assays we investigated whether PKRi affects any other protein kinase. These analyses demonstrated that PKRi has no major inhibitory effect on pro-apoptotic kinases such as the c-Jun N-terminal kinases, the p38 MAP kinases and the death-associated protein kinases, or on other kinases including c-Raf, MEK1, MKK6 and MKK7. PKRi does, however, inhibit the activity of certain cyclin-dependent kinases (CDKs), including CDK1, CDK2 and CDK5 both in vitro and in low potassium-treated neurons. Consistent with its inhibitory action on mitotic CDKs, the treatment of HT-22 and HEK293T cell lines with PKRi sharply reduces the rate of cell cycle progression. Taken together with the established role of CDK activation in the promotion of neurodegeneration, our results suggest that PKRi exerts its neuroprotective action by inhibiting CDK.