In vitro putative cytochrome P450 3A mediated activity, and inhibition thereof, were measured in four avian species using midazolam (MDZ) as a substrate and ketoconazole as an inhibitor. All species produced 1-hydroxymidazolam (1-OH MDZ) to a much greater extent than 4-hydroxymidazolam (4-OH MDZ). Calculated Vmaxapparent values for formation of 1-OH MDZ were 117+/-17, 239+/-108, 437+/-168, and 201+/-55 pmol/mg protein*min and Kmapparent values were 2.1+/-0.8, 2.4+/-1.6, 6.7+/-5.1 and 3.2+/-2.1 microm for chicken, turkey, pheasant and bobwhite quail, respectively. For the formation of 4-OH MDZ the Vmaxapparent values were 21+/-10, 94+/-46, 144+/-112, and 68+/-30 pmol/mg protein*min and Kmapparent values for 4-OH MDZ formation were 12.4+/-10.1, 18.0+/-10.8, 38.6+/-34.7 and 29.1+/-10.1 microm for chicken, turkey, pheasant and bobwhite quail, respectively. In all four species, ketoconazole inhibited the production of both major metabolites of MDZ, with 4-OH MDZ formation more sensitive to inhibition than 1-OH MDZ. Pheasant and bobwhite quail appeared most sensitive to ketoconazole inhibition.