We studied the properties of the ectokinase activity on the outer cell surfaces of RBL-2H3 cells and examined the phosphorylation of exogenous substrates to clarify the substrate specificity of the ectokinases on RBL-2H3 cells. Among the several protein substrates tested, casein was the most strongly phosphorylated with [gamma-32P]ATP, and the net incorporation of 32P into casein was 0.65 pmol P/50 microg/10(6) cells. Casein kinase II peptide was also phosphorylated with [gamma-32P]ATP. The phosphorylation of casein and casein kinase II peptide was almost completely inhibited by the addition of 3 microg/ml of cell-impermeable K252b. Phosphorylation of casein and casein kinase II peptide was also observed by [gamma-32P]GTP. Western blot analysis using anti-casein kinase II antibody revealed a 44-kDa casein kinase band in the membrane fraction and Fc epsilonRI complexes. The immunofluorescence microscopic analysis using anti-casein kinase II antibody showed the existence of casein kinase II on the surface of the cells. This is the first report about the existence of ectokinase on mast cells.