It has been proposed that coumarin and indandione anticoagulants inhibit clotting protein synthesis by preventing the regeneration of vitamin K1 from its 2,3-epoxide metabolic. To test this hypothesis, the effects of nine coumarin and indandiones on prothrombin synthesis and the conversion of vitamin K1 epoxide to vitamin K1 were measured. There was a good correlation between the inhibition of prothrombin synthesis and the epoxide-K1 conversion both in vivo and in vitro. Diphenadione (2-diphenyl acetyl-1,3-indandione) caused a delayed inhibition of prothrombin synthesis which correlated with a delayed inhibition of the epoxide-K1 conversion in vivo. These results provide strong evidence for the proposed mechanism of action of coumarin and indandione anticoagulants.