1. The study aimed to ascertain the enzyme kinetic basis for breed differences in the biotransformation of propofol in dog and to identify the responsible canine cytochrome P450 (CYP) isoenzymes. 2. The NADPH-dependent formation of 4-hydroxypropofol (the rate-limiting biotransformation in dog) was assayed using hepatic microsomes from the male greyhound and beagle, and from both sexes in mixed-breed dogs (five of each). 3. Enzyme kinetic analysis revealed that whereas there were no significant differences in Km, Vmax averaged > 3-fold lower in greyhound compared with beagle (p = 0.032). Although average Vmax was > 3-fold higher in the male compared with female mixed-breed dogs, this difference did not achieve statistical significance (p = 0.095), probably because of the high variability of data from mixed-breed dogs. 4. Chloramphenicol (a specific CYP2B11 inhibitor) and diethyldithiocarbamate (a non-specific CYP2 inhibitor) inhibited propofol hydroxylation in all microsomes. Quinine (a CYP2D15 inhibitor) was also inhibitory, but only in one-half of the microsomes examined. Immuno-inhibition by anti-CYP2B1 sera resulted in > 50% reduction in metabolite formation in all dogs except mixed-breed females, which showed a 30% reduction. Differences in propofol hydroxylase activity between microsomal preparations were primarily attributed to a component that was sensitive to inhibition by chloramphenicol and anti-CYP2B1 sera. 5. The results indicate that propofol hydroxylation in dog is primarily mediated by CYP2B11 and that breed (and possibly gender) differences in propofol metabolism may result from differences in the liver content of this CYP.