The substitution of lanthanide ions for Ca(II) in the Ca(II)-binding sites of prothrombin and the derivatives of prothrombin activation and in the metal-dependent conversion of prothrombin or prethrombin 1 to thrombin was studied at pH 6.8. Gd(III), Tb(III), La(III), Dy(III), Pr(III), Sm(III), and Ce(III) may be substituted for Ca(II) in the generation of thrombin from prothrombin or prethrombin 1 by activated factor X. The rates of thrombin generation in the presence of optimal concentrations of Gd(III) were about 25% for prothrombin and prethrombin 1 compared to the rate of thrombin generation with optimal concentrations of Ca(II). Maximal rates of thrombin generation were observed at 20 muM Gd(III) using prothrombin as substrate, compared to 10 muM Gd(III) when prethrombin 1 was employed. Using the steady state rate-dialysis method, the high affinity metal-binding sites of prothrombin and the products formed during prothrombin activation were characterized using 153Gd(III). Prothrombin has two high affinity binding sites for Gd(III) (Kd = 0.75 muM). Prethrombin 1 and prethrombin 2 each bind one Gd(III) tightly (Kd = 1.10 muM and 0.81 muM, respectively). Fragment 1, the phospholipid-binding portion of prothrombin, has two sites which bind Gd(III) tightly (Kd 0.16 muM). Fragment 2 has no high affinity metal-binding sites, but has intermediate affinity metal-binding sites (Kd greater than 1.6 muM). Thrombin has numerous high affinity binding sites (Kd less than 0.1 muM), suggesting that the conversion of prethrombin 2 to thrombin is associated with a significant change in tertiary structure. These results indicate that Gd(III) binds tightly to the metal-binding sites of these proteins and can substitute for Ca(II) in metal-dependent prothrombin activation. In the activation of prothrombin by activated factor X, these data suggest that Ca(II) is required for metal-dependent factor V and phospholipid binding and not as a cofactor in enzyme catalysis.