The glucuronidation of morphine, naloxone, and buprenorphine by the liver and intestine has been assessed both in vitro and in vivo in the rat. Using microsomes, Vmax/Km ratios were estimated as measures of intrinsic enzyme activity, and a wide range of ratios were obtained (500-fold). The ratio for the intestine was consistently less than for the liver, and the rank order of activities within each tissue was morphine less than naloxone less than buprenorphine. Using various routes of administration, plasma concentration-time profiles for each compound were determined and used to estimate hepatic and intestinal extraction ratios. These extraction ratios were dose-independent and more substantial for the liver than the intestine. For each tissue, buprenorphine and naloxone showed similar extraction, whereas morphine was less. Plasma binding and blood/plasma concentration ratios were determined, and perfusion models were used to calculate intrinsic clearance. This in vivo parameter of enzyme activity showed a wide range (200-fold), comparable to the spread of microsomal Vmax/Km ratios. Although the in vivo parameters consistently gave measures 10- to 30-fold higher than the in vitro parameters, the rank order was identical for the two sets. These data suggest that a comparative approach to correlate in vitro and in vivo data has advantages over the use of absolute drug characteristics.