Prothrombin activation by prothrombinase was investigated on platelets adhered onto a fibrinogen-coated coverslip mounted in a flow chamber. Once bound to the fibrinogen, platelets gradually changed their morphology: they developed pseudopods, spread over the surface and finally transformed into balloon-shaped cells. This last morphologic change required the presence of calcium and was accompanied by the exposure of procoagulant phospholipid at the outer membrane as detected by the capability of the platelets to bind fluorescein-labelled annexin V. Prothrombinase complexes were allowed to assemble on these adhered platelets by perfusion with factor Xa and varying concentrations of factor Va and prothrombin. The steady-state rate of thrombin formation during continuous flow increased with the prothrombin concentration but not with the factor Va between 0.05 and 0.5 nM. Once prothrombinase was assembled, factor Xa could be omitted from the perfusion mixture without affecting the steady state rate of thrombin production. Our study demonstrates the efficient ability of the procoagulant surface of adherent platelet to support the assembly of stable prothrombinase complexes. Thrombin production was limited by the rate of supply of prothrombin towards the catalytic surface.