Laromustine (VNP40101M; 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino) carbonylhydrazine) is a novel sulfonylhydrazine alkylating agent. Phase 1 metabolism of laromustine was reported recently and showed that laromustine undergoes rearrangement, dehalogenation, and hydrolysis at physiological pH to form active moieties. (1) A mechanism for the rearrangement was proposed on the basis of fragmentation ions. (1) (,) (2) In this article, we report the phase II conjugates of VNP40101M and VNP4090CE which were formed after incubation of VNP40101M or VNP4090CE with pooled human liver microsomes (HLM) and cofactors nicotinamide adenine dinucleotide phosphate (NADPH), glutathione (GSH), N-acetylecysteine (NAC), and cysteine (CYS). Eight novel phase II conjugates (M-1 to M-8) were identified and characterized by hydrogen-deuterium exchange (H-D), stable isotope ((13)C-labeled VNP40101M), and MS(n) experiments. M-4 and M-5 were further confirmed by nuclear magnetic resonance spectroscopy (NMR). The short-lived CH(3)SO(2)CH(2)CH(2)-, methylformamide and CH(3)SO(2)NHN═CHCH(2)- moieties were generated from VNP40101M. The reactive intermediates CH(3)SO(2)CH(2)CH(2)- and methylformamide formed conjugates with GSH, CYS, and NAC. The CH(3)SO(2)NHN═CHCH(2)- moiety formed conjugates with GSH and NAC. M-2, M-4, and M-6 were only detected from the incubation of VNP40101M because VNP4090CE does not contain a methylformamide group. All other conjugates were formed by both VNP40101M and VNP4090CE. The in vitro studies found that VNP40101M and VNP4090CE undergo activation in human liver microsomes. The results from this study showed that laromustine produces several reactive intermediates that may play a role in the toxicities seen in the clinical trials.