Steady state kinetic studies have been performed to investigate the formation of thrombin from prothrombin by human coagulation Factor Xa in the presence of Ca2+ and phospholipid. The concentration of ligand which gives 50% of the maximum velocity (K0.5) is 2.3 mM for Ca2+, 7.4 microM for phospholipid, and 0.006 microM for prothrombin. Hill plots of the Ca2+ enhancement of the reaction give a Hill coefficient of 3.1, indicating positive cooperativity. The initial velocity patterns are consistent with an ordered addition of reactants with phospholipid as the second reactant to bind to the enzyme. Although our results do not differentiate between Ca2+ or the prothrombin substrate as the first reactant to bind to Factor Xa, it is established that Ca2+ can bind to Factor Xa in the absence of the other reactants. Thus, the most probable order of addition of reactants is Ca2+, phospholipid, and the prothrombin substrate. Plots of (v)-1 versus (prothrombin)-1 or (v)-1 versus [(Ca2+)3]-1 at several constant concentrations of phospholipid indicate that the major effect of phospholipid is to increase the turnover number of Factor Xa.