Detergent-permeabilized EGFR-T17 fibroblasts, which overexpress the human epidermal growth factor (EGF) receptor, phosphorylate both poly-L-(glutamic acid, tyrosine) and exogenous calmodulin in an EGF-stimulated manner. Phosphorylation of calmodulin requires the presence of cationic polypeptides, such as poly-L-(lysine) or histones, which exert a biphasic effect toward calmodulin phosphorylation. Optimum cationic polypeptide/calmodulin molar ratios of 0.3 and 7 were determined for poly-L-(lysine) and histones, respectively. Maximum levels of calmodulin phosphorylation were attained in the absence of free calcium, and a strong inhibition of this process was observed at very low concentrations (Ki = 0.2 microM) of this cation. The incorporation of phosphate into calmodulin occurred predominantly on tyrosine residue(s) and was stimulated 34-fold by EGF.