A full-length cDNA encoding for a microsomal juvenile hormone (JH)-metabolizing epoxide hydrolase (TmEH-1) was isolated from a cDNA library constructed from fat body of last stadium (wandering) cabbage loopers, Trichoplusia ni, at the exact developmental time of maximum JH epoxide hydrolase activity. TmEH-1 was 1887 base pairs in length with a 1389 base pair open reading frame encoding 463 amino acids. Amino acid sequence analysis showed that TmEH-1 was most similar to and contained the exact catalytic triad (Asp-226, Glu-403 and His-430) found in microsomal epoxide hydrolases. TmEH-1-specific message was present along with JH III epoxide hydrolase activity in fat body in feeding (days 1 and 2) and wandering (day 3) larvae with the peak in message level preceding the peak in JH epoxide hydrolase activity by 1 day. When TmEH-1 was expressed in baculovirus-infected Spodoptera frugiperda cells, a 46,000 molecular weight protein appeared on SDS-PAGE which corresponded to the predicted size coded by the TmEH-1 message and which was positively correlated with increases in JH III epoxide hydrolase activity above that of wild-type controls. In subcellular distribution studies, 58% of the juvenile hormone III epoxide hydrolase activity was in the insoluble fractions. Baculovirus expressed TmEH-1 demonstrated a higher specific activity for JH III as compared to the general EH substrates, cis- and trans-stilbene oxide. Southern blot analyses suggested that multiple epoxide hydrolase genes are present in T. ni.