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 Km and kcat 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 Ki of 42 μM and a maximum rate of inactivation of 0.29 min-1. A partition ratio (kcat/kinact) was calculated to be 0.06.