Usingin vitrotechniques, the present study demonstrates that CYP2D6, 2C19 and 3A4 are involved in N-demethylation of citalopram (CIT) enantiomers. Human liver microsome incubations performed with specific inhibitors of these three CYP isozymes have shown up to 60% inhibition of demethylcitalopram production. cDNA expressed human cytochrome P-450 3A4, 2C19 and 2D6 isozymes, but not CYP1A2, were identified to be involved in N-demethylation of CIT enantiomers. Kinetics using cDNA expressed CYP2C19 and CYP3A4 show Kmvalues in the same range: 198 µM, 211 µM for CYP2C19 and 169 µM, 163 µM for CYP3A4 for S- and R-CIT demethylation, respectively. In contrast, kinetics using cDNA expressed CYP 2D6 show a Kmof 18 µM and 22 µM for S- and R-CIT demethylation, respectively. Nevertheless, kinetics using cDNA expressed CYP2C19 and 3A4 have a range of Vmaxvalues ten times higher than that of CYP2D6. For this reason, intrinsic clearance values (Vmax/Km) for Sand R-CIT were within a small range for these three isozymes: 0.25 to 0.39 µlh-1x pmol-1of CYP. CYP2D6 has an opposite stereoselectivity in the biotransformation of CIT enantiomers than CYP2C19 and 3A4; the S/R ratios of the intrinsic clearance were 0.71, 1.57 and 1.37, respectively. Taking into account that CYP isozymes are expressed at various levels, CYP2D6, which is expressed at lower levels than CYP2C19 and CYP3A4, plays a minor role in the biotransformation of CIT enantiomers. These results confirm that the use of cDNA expressed CYP isozymes is a potent tool for the measurement of kinetic constants and help to predict clearance modifications of CIT enantiomers, especially in poor metabolizers of mephenytoin (with a CYP2C19 deficiency) or patients comedicated with potent CYP2C19 or 3A4 inhibitor(s). For instance, fluvoxamine (100µM) inhibits CIT N-demethylation by 64% in microsomes.