Studies were carried out at pH 7.0 and Γ/2 0.15 before addition of CaCl 2 or EDTA. Clotting time, τ, at 3.03 μ m fibrinogen and 0.91 u/ml thrombin was determined for equilibrium systems. With added Ca 2+, τ decreases, from τ 0 at 0 added Ca 2+ (mean, 29.7 ± 3 s), by ~3 s at 5 m m added Ca 2+. With added EDTA, τ increases sigmoidally from τ 0 at 0 EDTA to a maximum (mean τ m = 142 ± 23 s) at ~200 μ m EDTA. τ then decreases slightly to a minimum at ~1.3 m m and finally increases to infinity at ~10 m m EDTA. Between 0 and 1.3 m m EDTA, effects on clotting time are completely reversed by adding Ca 2+ and, after equilibration at 400 μ m EDTA, τ is independent of EDTA concentration. Thus, up to 400 μ m EDTA, effects on clotting time are attributed to decreasing fibrinogen bound Ca 2+. Between 5 m m Ca 2+ and 200 μ m EDTA it is assumed that an equilibrium distribution of fibrinogen species having 3, 2, 1, or 0 bound calcium ions is established and that a clotting time is determined by the sum of products of species fractional abundance and pure species clotting time. Analysis indicates that pure species clotting times increase proportionately with decreasing Ca 2+ binding, binding sites are nearly independent, and the microscopic association constant for the first bound Ca 2+ is ~4.9 × 10 m −1. Effects of adding Ca 2+ at times t 1 after thrombin addition to systems initially equilibrated at 200 μ m EDTA were determined. Analysis of the relation between τ and t 1 indicates that as Ca 2+ binding decreases, rate constants for release of B peptides decrease less than those for release of A peptides. As EDTA concentration is increased above 1.3 m m, inhibitory effects of EDTA and CaEDTA progressively increase.