Previous studies have suggested that human platelets can promote the activation of factor XI by two different mechanisms, one requiring factor XII and ADP-treated platelets and the other requiring collagen-treated platelets in the apparent absence of factor XII. To investigate these hypotheses, isolated platelets were tested for their capacity to promote the activation and cleavage of purified factors XII and XI in various mixtures of purified factor XII, kallikrein, high molecular weight (HMW) kininogen, and factor XI. The activation of factor XII or factor XI was tested in clotting assays using plasmas deficient in factor XII, XI, IX, or VII and the limited proteolytic cleavage of 125l-labeled factor XII or XI was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence of reducing agents. That ADP- or collagen-treated platelets can promote the proteolytic activation of factor XII in mixtures containing kallikrein and HMW kininogen was shown by: (1) the proteolytic cleavage of factor XII; (2) the development of factor XII. coagulant activity; and (3) the proteolytic cleavage of 125l-factor XI. Platelets treated with collagen or thrombin were shown by both coagulant assays and by cleavage studies to participate with HMW kininogen and kallikrein in the proteolytic activation of factor XI by mechanisms that were partially dependent upon and partially independent of factor XII. That these zymogen activations are platelet-related was demonstrated by: (1) the enhanced coagulant activity and proteolysis observed after platelet stimulation with ADP, collagen or thrombin; (2) the inhibition of proteolytic activation seen when platelet responses were inhibited by indomethacin; (3) the absence of proteolytic activation in the absence of platelets or other surfaces; and (4) the localization of cleavage products of factor XI in platelet pellets. Experiments with celite or with unstimulated platelets present showed a linear relationship between factor-XII coagulant activity and proteolysis of factor XII. However, with activated platelets present the coagulant activity was two to fourfold greater than that observed for an equivalent amount of factor XII cleaved with celite present. These studies demonstrate that platelets can promote the proteolytic activation of factor XII by kallikrein and of factor XI by both factor-XII dependent and factor-XII-independent mechanisms.