A microsomal cytochrome P450 from a cell line of the insect Chironomus tentans has been shown to hydroxylate the steroid hormone 20-hydroxyecdysone at C(26) to yield 20,26-dihydroxyecdysone, P1, which is further metabolized to P2 and P3. Based on 1H NMR studies, acetonide formation and quantum chemical calculations, P2 and P3 represent novel slowly interconvertible geometrical isomers, occurring at a 3:1 ratio, presumably arising from hemiacetal formation between the 26-aldehyde group and the 22 R-hydroxyl group to build a tetrahydropyran ring in the side chain. The stereochemistry at C(26) was S in P2 ( trans-diol) and R in P3 ( cis-diol), respectively. Both metabolites showed S configuration at C(25). With Chironomus cells, P2/ P3 was inactive as both a hormonal agonist and antagonist, whereas 20,26-dihydroxyecdysone ( P1) showed weak agonist activity. Thus, cytochrome P450-mediated inactivation of 20-hydroxyecdysone may be responsible for the hormonal insensitivity observed in some subclones of this cell line.