The activation rate of prothrombin and its conversion into thrombin through a proteolytic degradation is enhanced by phospholipids in the presence of Ca++. In this work, we could elucidate stoechiometric and structural aspects of the interaction between prothrombin, phospholipids and Ca++. We studied the adsorption eguilibria of Ca++and of prothrombin on phospholipids monolayers by a new approach which combines two complementary methods of surface measurement.Electrochemical investigations on lipid monolayer interacting with prothromoin showed penetration of prothrombin in presence or not of Ca++(by increasing the lipid layer capacitance), with a preferential orientation. The adsorption of Ca++and prothrombin to the lipid layer was measured by counting the surface radioactivity of 45Ca and 3H-prothrombin. Synergetic attachment of Ca++and prothrombin to the phospholipid water interface was observed and the number of Ca++per attached prothrombin was determined. The two methods give comparative results for the proposed ionic-hydrophobic interaction.