A glutathione S-transferase (GST) with activity toward 1,2-epoxy-2-methyl-3-butene (isoprene monoxide) and cis-1,2-dichloroepoxyethane was purified from the isoprene-utilizing bacterium Rhodococcus sp. strain AD45. The homodimeric enzyme (two subunits of 27 kDa each) catalyzed the glutathione (GSH)-dependent ring opening of various epoxides. At 5 mM GSH, the enzyme followed Michaelis-Menten kinetics for isoprene monoxide and cis-1,2-dichloroepoxyethane, with V_max values of 66 and 2.4 µmol min¹ mg of protein¹ and K_m values of 0.3 and 0.1 mM for isoprene monoxide and cis-1,2-dichloroepoxyethane, respectively. Activities increased linearly with the GSH concentration up to 25 mM. ¹H nuclear magnetic resonance spectroscopy showed that the product of GSH conjugation to isoprene monoxide was 1-hydroxy-2-glutathionyl-2-methyl-3-butene (HGMB). Thus, nucleophilic attack of GSH occurred on the tertiary carbon atom of the epoxide ring. HGMB was further converted by an NAD⁺-dependent dehydrogenase, and this enzyme was also purified from isoprene-grown cells. The homodimeric enzyme (two subunits of 25 kDa each) showed a high activity for HGMB, whereas simple primary and secondary alcohols were not oxidized. The enzyme catalyzed the sequential oxidation of the alcohol function to the corresponding aldehyde and carboxylic acid and followed Michaelis-Menten kinetics with respect to NAD⁺ and HGMB. The results suggest that the initial steps in isoprene metabolism are a monooxygenase-catalyzed conversion to isoprene monoxide, a GST-catalyzed conjugation to HGMB, and a dehydrogenase-catalyzed two-step oxidation to 2-glutathionyl-2-methyl-3-butenoic acid.