Androgens (androsterone, dihydrotestosterone and testosterone) and estrogens (estradiol, estriol and estrone) were incubated with liver microsomes from rats, dogs, monkeys and humans in the presence of uridine diphosphoglucuronic acid (UDPGA), and the glucuronides produced were structurally characterized by liquid chromatography-tandem mass spectrometry. After 2-h incubation with dog liver microsomes, all substrates tested were converted (approximately 2-10%) to structurally novel diglucuronides, where two glucuronosyl groups are bound to a single hydroxyl group in tandem. Two-dimensional nuclear magnetic resonance spectroscopy unambiguously elucidated the chemical structures of the 3-O-diglucuronide of estrone and the 17-O-diglucuronide of testosterone isolated from the incubation mixture. Monkey and human liver microsomes were also found to have the activity to form this type of diglucuronide, albeit more slowly than the dog liver microsomes, but rat liver microsomes produced no detectable diglucuronides. The rate of formation of estrone 3-O-diglucuronide from the corresponding monoglucuronide in dog liver microsomes followed classical Michaelis-Menten kinetics at substrate concentrations from 50 to 1000 microM, with a K(m) value of 127.1 microM and a V(max) value of 47.0 pmol/min/mg protein.