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Use of the reductant dithiothreitol (DTT) as a substrate for measuring vitamin K 2,3-epoxide reductase (VKOR) activity in vitro has been reported to be problematic because it enables side reactions involving the vitamin K 1 2,3-epoxide (K 1 >O) substrate. Here we characterize specific problems when using DTT and show that tris(3-hydroxypropyl)phosphine (THPP) is a reliable alternative to DTT for in vitro assessment of VKOR enzymatic activity. In addition, the pH buffering compound imidazole was found to be problematic in enhancing DTT-dependent non-enzymatic side reactions. Using THPP and phosphate-based pH buffering, we measured apparent Michaelis–Menten constants of 1.20 μM for K 1 >O and 260 μM for the active neutral form of THPP. The K m value for K 1 >O is in agreement with the value that we previously obtained using DTT (1.24 μM). Using THPP, we successfully eliminated non-enzymatic production of 3-hydroxyvitamin K 1 and its previously reported base-catalyzed conversion to K 1 , both of which were shown to occur when DTT and imidazole are used as the reductant and pH buffer, respectively, in the in vitro VKOR assay. Accordingly, substitution of THPP for DTT in the in vitro VKOR assay will ensure more accurate enzymatic measurements and assessment of warfarin and other 4-hydroxycoumarin inhibition constants. [ABSTRACT FROM AUTHOR]