AbstractIn a study designed to examine the nature of short‐lived, electrophilic intermediates liberated during decomposition ofN,N′‐bis(2‐chloroethyl)‐N‐nitrosourea (BCNU)in vitroand also on administration of BCNU (140 μmol i.p.) to ratsin vivo, both on‐line and off‐line LC/MS/MS techniques were employed to detect and characterize the corresponding glutathione (GSH) adducts present in incubation media and excreted into bile, respectively.In vitro, four GSH conjugates were formed and these were identified, on the basis of their product ion spectra, as products ofS‐andN‐carbamoylation and alkylation reactions. Although the relative proportions of thesein vitroadducts were found to depend on the molar ratios of GSH and BCNU, the major adduct under all conditions studied proved to beS‐(2‐chloroethylcarbamoyl)glutathione (SCG). Analysis of untreated bile samples by means of on‐line LC/MS/MS with constant neutral loss (129 u) and precursor ion (m/z179) scanning techniques again led to the detection of four GSH conjugates, although only one of these (SCG) was common to the group of adducts identifiedin vitro.All of the GSH conjugates detected in bile represented products ofS‐carbamoylation, indicating that the alkylating moiety released from BCNU undergoes reactionsin vivowith