Pretreatment of hepatocytes with Ca2+ and A23187 resulted in the decrease in epidermal growth factor (EGF) binding to the receptor. This decrease could be induced by only the pretreatment of the isolated liver membrane with Ca2+. By the pretreatment with 1 mM Ca2+ for 30 min, EGF binding was decreased to 40% of control. Scatchard plot analyses indicated that high affinity binding sites of EGF receptor disappeared after Ca2+ pretreatment. No effect of Ca2+ pretreatment on the degradation of receptor occurred. The Ca2+ effect was prevented by monodansylcadaverine or iodoacetamide, transglutaminase (TGase) inhibitors. After membrane-bound TGase was inactivated, the Ca2+ effect did not occur, except in the presence of purified TGase. Labeled putrescine was incorporated into some proteins of membrane with Ca2+ treatment. These results suggest that Ca(2+)-activated TGase induces the decrease in EGF binding to the receptor via modification of some membrane protein.