Hepatic monoxygenases metabolize steroids that in turn influence reproduction. Experiments were conducted to establish the effect of dose of phenobarbital and level of dietary protein on hepatic monoxygenase. In Exp. 1, ewes were given either 0, .5, 1, 2 or 3 g phenobarbital (PB) daily for 4 to 8 d. After 48 h after last dose of PB, cytochrome P-450 was higher (P less than .01) in all ewes given PB than in controls and was higher (P less than .01) in ewes given PB for 8 d than in ewes treated for 4 d. In Exp. 2, either 0 or 1 g of PB was given orally for 8 d. Liver samples were collected 1, 4, 7 or 10 d after last treatment. Both cytochrome P-450 (P less than .01) and nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome c reductase (P less than .06) were higher in ewes receiving PB than in controls. Cytochrome P-450 was twice as high in treated ewes as in control ewes on the 8th d of treatment, but concentrations returned to control levels 10 d after last treatment. Microsomal protein and cytochrome b5 were not affected by PB (P greater than .10). Ewes in Exp. 3 receiving a diet containing 14.8% crude protein for 10 d had higher levels of cytochrome P-450 (P less than .01) than ewes fed 4.7% crude protein. Protein did not affect microsomal protein or NADPH cytochrome c reductase. These data suggest that a relationship among PB, dietary protein and monoxygenases exists and provide information that will lead to a better understanding of the relationship between diet and reproduction.