The guanine nucleotide exchange factor (GEF) is a multi-subunit protein which catalyzes the exchange of GDP for GTP in eukaryotic chain initiation factor 2. Phosphorylation of the 82-kDa subunit of GEF in vitro by casein kinase II (CK-II) is associated with a 5-fold increase in nucleotide exchange activity. However, phosphorylation of GEF in vivo has not been studied, and the kinase(s) that phosphorylate GEF have not been identified. The 82-kDa subunit of GEF was partially sequenced, and a synthetic peptide was used to generate polyclonal anti-peptide antibodies that react specifically with this subunit. To examine the phosphorylation of GEF in intact cells, the protein was isolated and purified extensively from metabolically 32P-labeled rabbit reticulocytes. Only the 82-kDa subunit was found to be phosphorylated, and on Western blots the anti-peptide antisera reacted specifically with the labeled subunit. Phosphoamino acid analysis indicated that phosphorylation occurred exclusively on Ser residues. Digestion with cyanogen bromide of in vivo labeled protein and GEF phosphorylated in vitro by CK-II produced comparable phosphopeptide maps. However, additional phosphopeptide bands were also observed with GEF derived from intact cells. Sequence analysis obtained by Edman degradation of the phosphopeptides was compared with the deduced amino acid sequence of a cloned 82-kDa subunit of GEF [Bushman, J. L., Asuru, A. I., Matts, R. L., & Hinnenbusch, A. G. (1993) Mol. Cell. Biol. 13, 1920-1932]. Putative sites of phosphorylation were identified at Ser 703 and/or 704, which contain the sequence S(P)XXD, a CK-II consensus recognition motif.(ABSTRACT TRUNCATED AT 250 WORDS)