Phosphorylation of secretory proteins is an uncommon event. In this manuscript, the phosphorylation of human chorionic gonadotropin, a glycoprotein hormone secreted by the JAR choriocarcinoma cell line, is described. Labeling of JAR cells with 32PO4 indicates that both the intracellular and the secreted forms of the free alpha subunit are phosphorylated. Although the secreted alpha beta dimer also incorporates 32PO4, there is little detectable phosphorylation of the intracellular precursors of alpha beta dimer, suggesting that dimer phosphorylation occurs as a late event in post-translational processing. In addition, phorbol 12-myristate 13-acetate markedly stimulates the phosphorylation of both intracellular and secreted forms of free alpha subunit and to a lesser extent of secreted alpha beta dimer. In vitro assays, using homogenates of JAR cells as a source of protein kinase activity, indicate that the uncombined alpha subunit is preferentially phosphorylated. The phosphorylation sites are on serine and threonine residues in the alpha subunit.