The beta subunit of eukaryotic elongation factor 1 (EF-1) catalyzes the GDP/GTP exchange activity on EF-1 alpha. In these studies, two cDNAs for the beta subunit of EF-1 from rabbit are cloned and sequenced. The cDNAs consist of 808 and 798 bp and are identical except for the 5' leader sequences of 67 and 57 bp. Both cDNAs code for a protein of 225 amino acids. Using the pT7-7 expression vector, EF-1 beta was expressed in Escherichia coli and purified to apparent homogeneity by chromatography on DEAE-cellulose and FPLC on Superose 12 and Mono Q. EF-1 beta was highly phosphorylated by casein kinase II, with up to 1.3 mol of phosphate incorporated per mol protein. From microsequence analysis and manual Edman degradation, the majority of the phosphate was shown to be present in serine 106 in the peptide DLFGS106DDEEES112EEA. Serine 112 was also phosphorylated by casein kinase II, but to a lesser extent. Previously, little phosphorylation of the beta subunit by casein kinase II was observed in native EF-1 unless GDP was bound to the alpha subunit (Palen, E., Venema, R.C., Chang, Y-W.E. and Traugh, J.A. (1994) Biochemistry, 8515-8520). In contrast, purified recombinant EF-1 beta was highly and specifically phosphorylated by casein kinase II; GDP and polylysine had little effect on the rate of phosphorylation of the purified subunit.