Elongation factor 1 (EF-1) consists of four subunits: the alpha subunit catalyzes the GTP-dependent binding of aminoacyl-tRNA to ribosomes while the beta, gamma, and delta subunits catalyze GDP/GTP exchange on EF-1 alpha. Phosphorylation of the beta subunit of EF-1 from rabbit reticulocytes by casein kinase II was stimulated up to 22-fold by polylysine, while basic proteins or polyarginine enhanced phosphorylation to a lesser extent. When physiological components of protein synthesis were examined as potential modulators of phosphorylation, ribosomal subunits had no effect, tRNA and poly(U) inhibited the phosphotransferase reaction, and GDP stimulated the initial rate of phosphorylation of EF-1 beta up to 3.8-fold; the degree of stimulation could be correlated with the amount of alpha subunit present in EF-1. No stimulation was observed with other nucleotides. Phosphorylation of EF-1 beta was on serine, and two-dimensional phosphopeptide mapping showed a single tryptic phosphopeptide in the presence of GDP or polylysine; the peptide was identical to that obtained with EF-1 phosphorylated in reticulocytes incubated with [32P]orthophosphate. EF-1 delta was also phosphorylated by casein kinase II, but only in the presence of GDP. Kinetic data showed GDP stimulated phosphorylation by increasing the Vmax with both the beta and delta subunits. The GDP-dependent stimulation of phosphorylation was specific for EF-1 and was not observed with calmodulin, beta-casein B, or c-Myc.(ABSTRACT TRUNCATED AT 250 WORDS)