The acid-catalysed (perchloric acid) hydrolysis of 1,2-epoxybutene-3 (EB) and of 1,2:3,4-diepoxybutane (DEB), two reactive epoxide metabolites of 1,3-butadiene (BD), was determined based on reaction of unchanged epoxide with 1-nitro-4-pyridyl-benzene (NBP). Related to different epoxide concentrations, both reactions were of first order. Related to different proton concentrations, second-order rate constants were obtained (approximately 10 s-1 M-1 perchloric acid for EB; approximately 0.01 s-1 M-1 perchloric acid for DEB). These data show a much higher chemical stability of DEB compared to EB. Moreover, EB and DEB were reacted at pH 7.2 in 10 mM TRIS buffer with deoxyguanosine (dG), guanosine (G) or calf thymus DNA. The unreacted epoxides (EB or DEB) present in the incubation mixtures with time were determined by gas chromatography. Consistent with the results of the acid-catalysed hydrolysis, the second-order rate constants for reaction with dG, G or DNA were more than 10-fold higher with EB, compared to those with DEB.