Rat liver microsomal 4-hydroxycoumarin binding was studied by assaying specific [14C]warfarin binding. Microsomes of warfarin-sensitive rats contained about 40 pmole of specific binding sites per mg of microsomal protein. There was no difference for R- or S-[14C]warfarin. Neither was there any difference between the enantiomers of acenocoumarol and phenprocoumon to prevent the in vitro racemic [14C]warfarin binding. Pretreatment of the microsomes with dithiothreitol, the in vitro reductor for microsomal vitamin K epoxide reductase activity, reduced the warfarin binding. Vitamin K epoxide nor vitamin K affected the warfarin binding. Microsomes of the Welsh warfarin resistant genotype showed weak warfarin binding properties. The Scottish resistant variant, on the other hand, did not differ from sensitive microsomes. Warfarin binding was reduced in microsomes of rats to which S-warfarin was administered. The reduction in warfarin binding was linear with the inhibition of microsomal vitamin K epoxide reductase activity and was linear with the amount of S-warfarin present in the microsomes. The results show the microsomal 4-hydroxycoumarin binding to be related to the target enzyme vitamin K epoxide reductase.