Incubation of a ribosome-free extract of rabbit reticulocytes or rat liver with [gamma-32P]ATP and Ca2+ results in incorporation of 32P predominantly into a single polypeptide of Mr approximately 100,000. This polypeptide is identified as elongation factor 2 (EF-2). Phosphorylation of EF-2 is strictly Ca2+-dependent and can be inhibited by the calmodulin antagonist trifluoperazine. It is suggested that the Ca2+/calmodulin-dependent phosphorylation of EF-2 is involved in regulation of protein biosynthesis.