A rapid and one time determinable TLC method for the monohydroxytestosterones formed by microsomal cytochrome P-450(P-450) was established, and applying this method the activity of P-450-dependent testosterone hydroxylases was observed in liver microsomes of rats pretreated with phenobarbital(PB). The 14 testosterone metabolites were found on the TLC plate. The amounts of 6-monohydroxytestosterones ($2{alpha}-$, $2{eta}-$, $6{alpha}-$, $6{eta}$, $-7{alpha}$-and $16{alpha}$-OH-T) were determined. The formation of these metabolites showed a good linearity(r = 0.9687-0.9999) corresponding to the concentration of P-450 added up to about 1.0 nmol/min/reaction mixture, and an excellent reproducibility (C.V.=$1.8{pm}1.0%$) when determined with 0.5 nmol P-450 in this assay system. The hydroxylation of testosterone to its $2{eta}$-OH-T by PB-inducible P-450 was increased (2 fold), while $2{alpha}-$ and $7{alpha}$-OH-T was rather decreased to 48 and 32%, respectively, compared with those of uninduced one. The $6{eta}$-OH-T was predominantly formed by uninducible and PB-inducible P-450, especially PB-inducible P-450 hydroxylated stereospecifically onto the C2-position of testosterone $(2{alpha}/2{eta}=1:3.8)$. However, the oxidation rates of testosterone to its $6{alpha}-$, $6{eta}-$ and $16{alpha}$-OH-T, and the total amount of testosterone metabolites identified were not changed significantly by the administration of PB.