Aims Nicotine is metabolised to cotinine (COT) and further to trans-3'-hydroxycotinine (HCOT) by the cytochrome P450 CYP2A6 enzyme. It has been demonstrated that the ratio of HCOT to COT is an index of CYP2A6 activity, thus a marker of nicotine metabolism, with higher ratios indicating faster metabolism. Evidence shows that the ratio, in urine, saliva or plasma, could predict responses to smoking cessation medication and guide pharmacotherapy. Since the literature about the association between this ratio and the cigarettes smoked per day (cpd) is not uniform, our aim was to investigate this relationship using urine samples from smoking females living in Romania, Portugal, Belgium and Slovenia. Methods Urine samples from mothers (aged up to 45 years) who participated in the EU project DEMOCOPHES(LIFE09/ENV/BE/00410) were used. The concentrations of COT and HCOT were determined by online-SPE UPLC MS/MS and subsequently normalised to creatinine content. The amount of cpd was available from the questionnaires. Results and conclusions As expected, a strong positive correlation between COT and HCOT levels was found. Regarding the association between HCOT/COT ratio and cpd, no correlation was observed when the data were globally considered. We confirmed that the correlation between cpd and cotinine differed according to the HCOT/COT ratio: higher correlations for slow metabolizers (low ratio) and almost no correlation for fast metabolizers (high ratio). A new analytical strategy is in progress to fully explore the usefulness of this ratio as biomarker of cigarette consumption.