This study is aimed to investigate the effect of the prolonged intake of conspicuous amounts of licorice (LE), or its natural constituent glycyrrhizin (G) on murine liver CYP-catalyzed drug metabolism. For this purpose the modulation of the regio- and stereo-selective hydroxylation of testosterone, together with the use of highly specific substrates as probes for different CYP isoforms such as ethoxyresorufin (CYP1A1), methoxyresorufin (1A2), pentoxyresorufin (2B1), p-nitrophenol (2E1) and aminopyrine (3A), were investigated. Daily doses of licorice root extract (3,138 or 6,276 mg/kg b.w. per os), or G (240 or 480 mg/kg b.w. per os), were administered to different groups of Swiss Albino CD1 mice of both sexes for 1, 4 or 10 consecutive days. While a single LE or G dose was unable to affect the multienzymatic CYP-system, using both schedules of repeated treatment, either LE or G were able to significantly induce hepatic CYP3A- and, to a lesser extent, 2B1- and 1A2-dependent microsomal monooxygenase activities, as well as 6beta- (mainly associated to CYP3A), 2alpha-, 6alpha- (CYP2A1, 2B1), 7alpha-, 16alpha- (CYP2B9) and 16beta-testosterone hydroxylase (TH) activities in male and female mice. Data on CYP3A modulation, the major isoform present in human liver, was confirmed by using Western immunoblotting with anti-CYP3A1/2 rabbit polyclonal antibodies raised against purified rat CYP3A. Northern blotting analysis using CYP3A cDNA biotinylated probe showed that the expression of such isozyme is regulated at the mRNA level. These results suggest that the induction of cytochrome P450-dependent activities by the prolonged intake of high LE or G doses, may result in accelerated metabolism of coadministered drugs with important implications for their disposition. The adverse effects associated with CYP changes such as toxicity/cotoxicity and comutagenicity may also have clinical consequences.