The catalytic subunit of cyclic AMP-dependent protein kinase (PKA) phosphorylated purified calcium/calmodulin-dependent eukaryotic elongation factor-2 (eEF-2) kinase, isolated from rabbit reticulocyte lysates. It maximally incorporated about 1 mol of phosphate/mol of eEF-2 kinase. The Km of eEF-2 kinase for PKA was calculated to be 7 microM. Phosphorylation of eEF-2 kinase by PKA induced calcium-independent activity which amounted to 40-50% of the total activity measured in the presence of calcium. Furthermore, the level of calcium-independent activity induced by phosphorylation by PKA was similar to that induced by the calcium-stimulated autophosphorylation of eEF-2 kinase. Phosphopeptide mapping of eEF-2 kinase labelled by autophosphorylation and by PKA revealed a number of common phosphopeptides. This suggests that PKA may phosphorylate the same site(s) which are phosphorylated autocatalytically and which are responsible for the induction of calcium-independent activity. The possible implications these findings have for the control of translation are discussed.