The spectral changes of cytochrome P-450 associated with mixed-function oxidation of hexobarbital and aminopyrine were investigated in perfused rat liver, using reflectance spectrophotometry. Simultaneously, the oxidation-reduction state of pyridine nucleotide(s) and oxygen uptake were measured from the same liver. Difference spectra were observed after infusion of hexobarbital or aminopyrine. Spectra obtained from livers of fasted, sodium phenobarbital-treated rats were similar to those of cytochrome P-450 . substrate complex (type I); the spectrum from fed, sodium phenobarbital-treated rats was a mixture of substrate . cytochrome P-450 complex and reduced (an oxygenated) cytochrome P-450. In fed, sodium phenobarbital-treated rats, the steady state level of reduced (an oxygenated) cytochrome P-450 was dependent on the concentration and the kind of substrates added, and the level correlated well with mixed-function oxidase activity. In the absence of exogenous substrates, the degree of the formation of reduced (an oxygenated) cytochrome P-450 was small. These results indicate that mixed-function oxidase activity in the intact cell is regulated by substrate-binding to cytochrome P-450. In fasted, sodium phenobarbital-treated rats, a substantial oxidation of pyridine nucleotide was observed in the presence of hexobarbital. The increase of oxygen uptake was a 2- to 2.5-fold smaller rate than in livers from fed, sodium phenobarbital-treated rats. Infusion of sorbitol (2 mM), a glycogenic substrate in fasted rats, stimulated oxygen uptake about 3-fold. Furthermore, reduced (an oxygenated) cytochrome P-450 increased in the presence of sorbitol. These results indicate that the rate of NADPH supply is rate-limiting for mixed-function oxidation in livers of fasted, sodium phenobarbital-treated rats. Finally, the difference spectrum of cytochrome P-450, the surface fluorescence of pyridine nucleotide, and the oxygen uptake were compared using livers from untreated, alloxan-treated and sodium phenobarbital-treated rats.