In a coagulation cascade, the conversion of the proenzyme prothrombin into the active enzyme thrombin occurs on the membrane of activated platelets. Since the kinetics of this conversion are considerably increased in the presence of negatively charged phospholipids, we investigated the modes of interaction of prothrombin(II) with such phospholipidic membranes. Since we have previously found that prothrombin penetrates into phospholipid monolayers and vesicles, in this work we studied which domains of this protein are involved in this penetration. Two complementary approaches were used with two complementary domains, fragment 1 (F1) and prethrombin 1 (P1). Penetration of the protein and its isolated and purified fragments into lipid monolayers with different phosphatidylserine contents was inferred from variations in the capacitance of this layer, measured by ac polarography. It was found that P1 and F1 penetrate in a cooperative manner and contribute in a synergic manner to the penetration of II, while prethrombin 2 (P2) has the highest tendency to penetrate alone. In the case of vesicles, the method is based on the photolabeling of the protein domains embedded in the membrane, using the apolar reagent 5- 125I-iodonaphthalene-1-azide. In the whole protein, P2 is the main penetrating domain, while P1 and F1 are weakly labeled. The results provide good evidence that the prethrombin 2 domain penetrates into both membrane models.