Orgaran (Org 10172), which has antithrombotic activity in man with apparently minimal bleeding side effects, is a mixture of low-molecular-weight heparan, dermatan, and chondroitin sulfates. The degrees to which the minimum concentrations of Orgaran, its fraction with high affinity for antithrombin III (Org 10849; AT III) and unfractionated heparin, which double the activated partial thromboplastin time (APTT) of pooled normal plasma, inhibit intrinsic activation of factor IX, factor X, and prothrombin were compared. Specific ELISAs were used to quantify the activation of each clotting factor. Factor IX activation, which began without a lag phase, preceded factor X and prothrombin activation by ∼ 15 and ∼ 25 s, respectively. When used at these functionally equivalent concentrations, heparin (2 µg/ml plasma), Orgaran (50 µg/ml plasma), and Org 10849 (20 µg/ml) could delay the onset of factor IX activation. Compared to control plasma, however, only Orgaran reduced the initial rate of factor IX activation. Heparin and Orgaran delayed the onset of factor X activation by 20 and 15 s, respectively, while Org 10849 could not delay the onset of factor X activation. In addition, each anticoagulant delayed the onset of prothrombin activation. Thus, at concentrations which double the APTT of normal plasma, the combined actions of heparan and dermatan sulfate present in Orgaran can apparently suppress factor IX activation more effectively than heparin, and delay the onset of factor X activation nearly as effectively as heparin. The coordinated inhibition of factor IX and factor X activation by Orgaran may contribute to its antithrombotic effectiveness.