The microstructure of polyisoprene synthesized with (BuCl)-Bu-t/TiCl4 initiating system is investigated using 1D and 2D (HSQC and HMBC) NMR spectroscopy. It is found that trans-1,4-units with regular (head-to-tail) and inverse (tail-totail) and (head-to-head) enchainments are predominant structures of unsaturated part of polymer chain, while 1,2-and 3,4-units are presented in minor amounts. The new methodology for the quantitative calculation of the content of different structural units in polyisoprene chain including both types of inverse trans-1,4-addition (tail-to-tail and head-to-head) is proposed. It is shown that head groups consist of tert-butyl group connected to trans-1,4-unit of polyisoprene chain. In addition, two types of chlorine-containing end groups are found (trans-4,1-Cl and 4,3-Cl), while conjugated double bonds at the chain end are totally absent. The methodology for the calculation of number-average functionality by tertbutyl head and chlorine end groups, respectively, is developed. (C) 2016 Wiley Periodicals, Inc.