Insulin caused a rapid, dose-dependent increase in the binding of 125I-insulin-like growth factor-II (IGF-II) to the surface of cultured H-35 hepatoma cells. The [32P]phosphate content of the IGF-II receptors, immunoprecipitated from extracts of H-35 cell monolayers previously incubated with [32P]phosphate for 24 h, was decreased after brief exposure of the cells to insulin. Analysis of tryptic digests of labeled IGF-II receptors by bidimensional peptide mapping revealed that the decrease in the content of [32P]phosphate occurred to varying degrees on three tryptic phosphopeptides. Thin layer electrophoresis of an acid hydrolysate of isolated IGF-II receptors revealed the presence of [32P] phosphoserine and [32P]phosphothreonine. Insulin treatment of cells caused a decrease in the labeled phosphoserine and phosphothreonine content of IGF-II receptors. The ability of a number of highly purified protein kinases (cAMP-dependent protein kinase, protein kinase C, phosphorylase kinase, and casein kinase II) to catalyze the phosphorylation of purified IGF-II receptors was examined. Casein kinase II was the only kinase capable of catalyzing the phosphorylation of the IGF-II receptor on serine and threonine residues under the conditions of our assay. Bidimensional peptide mapping revealed that the kinase catalyzed phosphorylation of the IGF-II receptor on a tryptic phosphopeptide which comigrated with the main tryptic phosphopeptide found in receptors obtained from cells labeled in vivo with [32P]phosphate. IGF-II receptors isolated by immunoadsorption from insulin-treated H-35 cells were phosphorylated in vitro by casein kinase II to a greater extent than the receptors isolated from control cells. Similarly, IGF-II receptors from plasma membranes obtained from insulin-treated adipocytes were phosphorylated by casein kinase II to a greater extent than the receptors from control adipocyte plasma membranes. Thus, the insulin-regulated phosphorylation sites on the IGF-II receptor appear to serve as substrates in vivo for casein kinase II or an enzyme with similar substrate specificity.