An isopentenol utilization pathway-based method for the investigation of the cyclization mechanism of terpene cyclases (TCs) is developed. By feeding deuterium-labeled prenols/isoprenols in combination with unlabeled ones to engineered E. coli hosts, terpene products with certain deuterium labeling patterns at hydrogen-bearing positions were obtained that can be used for deducing the cyclization processes, especially for those steps involving stereoselective hydride/proton shifts. Different types of TCs of varied origins for the biosynthesis of six known terpenes were used to test the scope and limitations of this method. Reliable results without significant deuterium dilution and scrambling are obtained by using this “deuterium-scanning” method and are consistent with those obtained previously. Limitations exist in the deuterium transfer process between those positions that are derived from the same labeled position of isoprenol, as exemplified by the failure of precisely tracking the origin of each deuterium in the labeled fusicocca-2,10(14)-diene obtained by feeding [2,2-2 H2]-isoprenol. Nonetheless, the newly developed method could be used as an alternate to those using custom-labeled oligoprenyl diphosphates for probing the cyclization mechanism of TCs.