The effect of two detergents, Triton X-100 and Brij 58, on the production rate of morphine-3-glucuronide by rat hepatic microsomes has been investigated over a range of detergent and substrate concentrations, using a specific HPLC assay. Activation of morphine-UDP-glucuronosyltransferase (morphine-UDPGT) by Triton X-100 was more complex than that shown by Brij 58. At the optimal concentration of Triton X-100 (0.1-0.125 mg Triton X-100/mg microsomal protein), relative metabolic activity (activity of morphine-UDPGT in the activated state/activity of morphine-UDPGT in the native state; RMA) was 0.9, 1.3 and 2.5 at morphine concentrations of 0.05, 0.5 and 2.5 mM, respectively. Analysis of results from six individual rats in the native and maximally activated state (0.125 mg Triton X-100/mg microsomal protein) showed that RMA was highly dependent upon substrate concentration (P < 0.0001). Activation produced by the optimal concentration of Brij 58 (0.15mg Brij 58/mg microsomal protein) was also dependent upon substrate concentration with values for RMA of 3.3, 6.4 and 9.3 at morphine concentrations of 0.05, 0.5 and 2.5 mM, respectively. Analysis of kinetic data is complicated by substrate concentration-dependent detergent activation. It is proposed that factors contributing to substrate concentration-dependent variable activation may include micellar solubilization of substrate by detergent and/or the presence of at least two enzyme forms capable of glucuronidating morphine with differential effects of detergents on these forms.