Phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2) is a well characterized mechanism regulating protein synthesis. Viral and cellular proteins have been identified that regulate the activity of the eIF-2alpha kinases. The regulatory protein, K3L, from vaccinia virus is homologous to the amino terminus of eIF-2alpha and is thought to inhibit the activity of the double-stranded RNA-dependent kinase suppressing the antiviral mechanism mediated by this kinase. We investigated whether K3L can inhibit the activity of the yeast eIF-2alpha kinase GCN2. Expression of K3L protein in yeast reduced the level of eIF-2alpha phosphorylation by GCN2 and blocked the stimulation of the general amino acid control pathway in response to starvation conditions. Accompanying in vitro studies showed that recombinant K3L protein reduced GCN2 autophosphorylation and phosphorylation eIF-2alpha. In agreement with the hypothesis that K3L inhibits eIF-2alpha kinases by functioning as a pseudosubstrate, we observed that K3L directly interacted with the kinase catalytic domain of GCN2. Together, these results indicate that K3L is a specific inhibitor of eIF-2alpha kinases from mammals and yeast and suggest that the kinases contain common structural features important for recognition of their substrate eIF-2alpha.