The inactivation of human factor XIa by human antithrombin III was studied under pseudo-first-order reaction conditions (excess antithrombin III) both in the absence and in the presence of heparin. The time course of inhibition was followed by using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. After electrophoresis, proteins were blotted onto nitrocellulose and stained either for glycoprotein or for antithrombin III using antibodies against antithrombin III. Concomitant with factor XIa inactivation, two new slower migrating bands, one of which represented the intermediate complex consisting of one antithrombin III complexed with factor XIa, appeared as a transient band. Complete inactivation resulted in a single band representing the complex of factor XIa with two antithrombin III molecules. Quantitative analysis of the time course of inactivation was accomplished by measurement of the disappearance of factor XIa amidolytic activity toward the chromogenic substrate S2366. Pseudo-first-order reaction kinetics were observed throughout. The rate constant of inactivation was found to be 10(3) M-1 s-1 in the absence of heparin and 26.7 X 10(3) M-1 s-1 in the presence of saturating amounts of heparin. From the kinetic data, a binding constant (Kd) of 0.14 microM was inferred for the binding of antithrombin III to heparin. The time course of inactivation and the distribution of the reaction products observed upon gel electrophoresis are best explained assuming a mechanism of inactivation in which the two active sites present in factor XIa are inhibited in random order (i.e., independent of each other) with the same rate constant of inhibition.