Mammalian cells contain a Ca2+/calmodulin-dependent protein kinase that specifically phosphorylates and inactivates elongation factor 2 (EF-2) in response to hormones and other agents which increase intracellular Ca2+ concentrations. Therefore, it has been proposed that the rate of translation in mammals is regulated by EF-2 phosphorylation. In the present study, EF-2 purified from the yeast Saccharomyces cerevisiae is shown to be a substrate for the mammalian EF-2 kinase. Furthermore, evidence was obtained using two-dimensional gel electrophoresis and peptide mapping which suggests that yeast EF-2 is a substrate for an endogenous kinase which phosphorylates the same site as the mammalian EF-2 kinase. Based on these findings, we propose that in yeast as in higher eukaryotes, the protein synthesis elongation cycle is regulated by phosphorylation of EF-2.