Although the formation of bilirubin monoglucuronide by hepatic microsomes has been easily demonstrable, that of bilirubin diglucuronide, the principal conjugate of bile, has been more difficult. Therefore, an examination of the uridine diphosphate glucuronate-dependent microsomal formation of these two conjugates has been made utilizing a high performance liquid chromatographic method which quantitates the isomeric forms of the products. Initial studies indicated that at high starting bilirubin concentrations, only bilirubin monoglucuronide was formed; whereas at lower concentrations (approximately 15 microM), bilirubin diglucuronide could be formed efficiently, but only under very specific conditions. Untreated microsomes and microsomes treated with Triton X-100 did not differ; each formed monoglucuronide efficiently, yet formed diglucuronide poorly. Digitonin or UDP-N-acetylglucosamine pretreatment, in contrast, was found to facilitate bilirubin diglucuronide formation, the former much more than the latter. The activity of mannose 6-phosphatase, an enzyme located on the inner surface of the microsomal vesicles, did not correlate well with the bilirubin diglucuronide formation. Time course studies with digitonin and UDP-N-acetylglucosamine indicated a precursor-product relation between bilirubin monoglucuronide and bilirubin diglucuronide, and product isomer composition studies indicated that the bilirubin tetrapyrroles were stable (no random dipyrrolic exchange had occurred). Temperature studies with the digitonin-treated preparation demonstrated an increase in monoglucuronide-forming activity over the 0-25 degrees C range, whereas diglucuronide formation increased dramatically over the range from 25 to 35 degrees C. The results indicate that microsomal diglucuronide-forming activity differs characteristically from monoglucuronide-forming activity, and that it is intensely sensitive to the manipulation of its microsomal membrane environment.