26,27-Dehydrocycloartenol (26,27-DHC) was shown to be a substrate for the soybean sterol methyltransferase (SMT) as well as a mechanism-based inhibitor of enzyme action. The K m and k c a t for 26,27-DHC was 10 μM and 0.018 min - 1 , respectively. SMT catalyzed 26,27-DHC to two products tentatively identified as 26-homocholesta-9,19-cyclo-23(24)E,26(26')-dienol and 26-homocholesta-9,19-cyclo-26(26')-en-3β,24β-diol by GC-MS. Inhibitor treatment was concentration- and time-dependent (pseudo-first-order kinetics). A replot of the half-lives for inactivation versus the inverse of the inactivator concentrations gave an apparent K i of 42 μM and a maximum rate of inactivation of 0.29 min - 1 . A partition ratio (k c a t /k i n a c t ) was calculated to be 0.06.