BACKGROUND AND OBJECTIVE: Mirtazapine is a tetracyclic antidepressant drug available as a racemic mixture of S(+)- and R(-)-mirtazapine. These enantiomers have different pharmacological properties, and both contribute to the clinical and adverse effects of the drug. Cytochrome P450 (CYP) 2D6 has been implicated in the metabolism of S(+)-mirtazapine. However, the effect of CYP2D6 on serum concentrations of the enantiomers of mirtazapine and its metabolites has not been assessed in patients on long-term treatment. The main objective of the study was to evaluate the effect of the CYP2D6 genotype on enantiomeric steady-state trough serum concentrations of mirtazapine and its metabolites N-desmethylmirtazapine and 8-hydroxymirtazapine. The effects of sex, age and smoking behaviour were also assessed. SUBJECTS AND METHODS: The study included 95 patients who had depression according to the Diagnostic and Statistical Manual of Mental Disorders - 4th Edition and were treated for 4 weeks with a daily dose of mirtazapine 30 mg. The serum concentrations of the enantiomers of mirtazapine and its metabolites were analysed by liquid chromatography-mass spectrometry, and the subjects were genotyped for CYP2D6 alleles *3, *4, *5 and *6 and gene duplication. RESULTS: Three subjects (3%) were classified as ultrarapid metabolizers (UMs), 56 (59%) as homozygous extensive metabolizers (EMs), 30 (32%) as heterozygous EMs and 6 (6%) as poor metabolizers (PMs) of CYP2D6. The median trough serum concentrations of S(+)-mirtazapine were higher in PMs (59 nmol/L, p = 0.016) and in heterozygous EMs (39 nmol/L, p = 0.013) than in homozygous EMs (28 nmol/L). PMs and heterozygous EMs also had higher mirtazapine S(+)/R(-) ratios (0.4) than homozygous EMs (0.3, p = 0.015 and 0.004, respectively). The S(+)-N-desmethylmirtazapine concentration was higher in PMs (16 nmol/L) than in homozygous EMs (7 nmol/L, p = 0.043). There was an association between the CYP2D6 genotype and the ratio between S(+)-8-hydroxymirtazapine and S(+)-mirtazapine, with a significantly higher ratio in homozygous EMs than in heterozygous EMs (0.11 vs 0.05, p = 0.007). The influence of the CYP2D6 genotype on S(+)-mirtazapine, the mirtazapine S(+)/R(-) ratio and S(+)-N-desmethylmirtazapine remained significant after correction for the influence of sex, age and smoking. Smokers had significantly lower concentrations of S(+)-mirtazapine (23 vs 39 nmol/L, p = 0.026) and R(-)-N-desmethylmirtazapine (39 vs 51 nmol/L, p = 0.036) and a significantly lower mirtazapine S(+)/R(-) ratio (0.28 vs 0.37, p = 0.014) than nonsmokers, and the effect of smoking remained significant after multivariate analysis. CONCLUSIONS: This study is the first to show the impact of the CYP2D6 genotype on steady-state serum concentrations of the enantiomers of mirtazapine and its metabolites. Our results also support the role of CYP1A2 in the metabolism of mirtazapine, with lower serum concentrations in smokers than in nonsmokers.