The effects of various polyunsaturated/saturated (P/S; 0.29-2.74) and n-6/n-3 (0.49-11.21) ratios of dietary fatty acids and of phenobarbital (PB) stimulation on the activity of liver microsomal mixed-function oxidase (MFO) system were studied in rats using a combination of palm oil, safflower oil and fish oil concentrate. When the n-6/n-3 ratio was kept constant (4.6-4.9) without induction by PB, the highest P/S ratio (2.74) tended to increase the basal PB-uninduced MFO activities; while the PB-induced MFO activities were elevated as the dietary P/S ratio increased. This definitely indicates the role of both n-6 and n-3 polyunsaturated fatty acids (PUFAs) in the elevation of MFO activities with and without PB stimulation. Whereas the n-6/n-3 ratio was varied while maintaining a constant P/S ratio (1.3), the PB-induced MFO activities were significantly elevated by decreasing the n-6/n-3 ratio although the MFO activities were not affected without PB stimulation. The MFO activities induced by PB were directly related to the n-3 PUFA content and inversely related to the n-6 PUFA content of the diets with n-6/n-3 ratios less than 4.55. These results obtained herein suggest that there are significant differences in the effects of n-6 and n-3 PUFAs on liver microsomal MFO activities, especially in relation to the induction of MFOs by PB.