Prothrombinase assembly was studied on macroscopic planar bilayers consisting of 20% dioleoyl-phosphatidylserine (DOPS) and 80% dioleoyl-phosphatidylcholine (DOPC). The dissociation constant for the binding of factor Xa to the bilayer, measured by ellipsometry, was K(d) = 47 +/- 8 nM (mean +/- S.D.) and this value was lowered to K(d) = 2.2 +/- 0.3 pM by preadsorption of factor Va. This latter value was determined from direct measurement of steady-state thrombin production. A comparable value of K(d) = 1.0 +/- 0.1 pM was found by repeating these experiments in suspensions of phospholipid vesicles, and it was verified that prothrombinase assembly was not influenced by the addition of prothrombin.Using a minute amount (0.094 fmol cm-2) of preadsorbed factor Va, it was found that the rate of prothrombinase assembly exceeds the rate of collisions between Xa molecules from the buffer and the sparse Va molecules on the bilayer. Apparently, factor Xa adsorbs first to the membrane and then associates rapidly with factor Va by lateral diffusion. The data indicate almost instantaneous equilibrium of this complex formation on the surface with a lower limit for the bimolecular rate constant of k(on) = 2.8 x 10(13) (mol/cm2)-1 s-1.In suspensions of small phospholipid vesicles, prothrombinase assembly is collisionally limited and the value of k(on) should be proportional to vesicle diameter. This was verified with a method for estimation of k(on) values from thrombin generation curves. Values of 0.36 x 10(9) and 1.6 x 10(9) M-1 s-1 were found for vesicles of 20-30- and 60-80-nm diameter, respectively.