The cdc2 protein kinase phosphorylates elongation factor-1 gamma (EF-1 gamma) during meiotic maturation of Xenopus oocytes. A synthetic peptide P2: PKKETPKKEKPA matching the cDNA-deduced sequence of EF-1 gamma was an in vitro substrate for cdc2 protein kinase and inhibited phosphorylation of EF-1 gamma. Tryptic hydrolysis of EF-1 gamma and the P2 peptide, both phosphorylated by cdc2 protein kinase, resulted in multiple partial digestion products generated by the presence of barely hydrolysable bonds. The two peptides obtained from the hydrolysis of EF-1 gamma comigrated exactly in two-dimensional separation with two of the P2 peptide hydrolysates. EF-1 gamma therefore contains one unique phosphoacceptor for cdc2 protein kinase, identified as threonine-230.