1. Rat liver microsomal metabolism of the enantiomers of warfarin and acenocoumarol (4'-nitrowarfarin) has been studied. The enantiomers of both compounds were hydroxylated mainly at the 6- and 7-positions. Acenocoumarol enantiomers were much better substrates for cytochromes P-450 than the corresponding warfarin enantiomers; Km values for the 6- and 7-hydroxylations were 2 to 19 times lower for R- and S-acenocoumarol than for warfarin. 2. Formation of the 6-, 7-, and 8-hydroxy-metabolites of warfarin was stereoselective for the R-enantiomer (the R/S ratio for total intrinsic clearance was about 3). 4'-Hydroxylation was not stereoselective. In contrast, formation of acenocoumarol metabolites was stereoselective for the S-enantiomer (the S/R ratio for total intrinsic clearance was about 3). 3. From the effects of phenobarbitone and methylcholanthrene induction, and inhibition by cimetidine, on in vitro metabolism of the enantiomers of both compounds, it was concluded that the differences between warfarin and acenocoumarol can be explained partly by the involvement of different enzymes.