1,4-14C Busulfan gave one main metabolite in the isolated perfused rat liver during 4 hr cyclic perfusion. The cumulative bile excretion contained about 38% of the total radioactivity. About 1% of unchanged 14C-busulfan was excreted in the bile. The metabolite was identified as gamma-glutamyl-beta-(S-tetrahydrothiophenium) alanyl-glycine (sulfonium ion of glutathione) by 252Cf-plasma desorption time-of-flight mass spectrometry. The formation of the metabolite was drastically decreased when the glutathione-S-transferase was inhibited, which indicates that the major reaction of busulfan with glutathione is enzymatic in nature. The sulfonium ion was more stable in the perfusate (t1/2 = 22.4 hr, 37 degrees C) than in the bile (t1/2 = 3.2 hr, 37 degrees C) at pH 7.4.