The aim of this study was to evaluate the self-reported and serum cotinine based prevalence rates of smoking among Iranian men, women and adolescents. The study was carried out on 2626 men and women aged 19 years and above and 836 high school students aged 14-18 years, randomly selected from different clusters and schools in Isfahan. The WHO smoking standard questionnaire was completed for all samples and serum cotinine level was measured using high-performance liquid chromatography in 10% and 20% of the original adult and adolescent samples, respectively. The prevalence of self-reported smoking among Iranian men and women aged 19 years and above was 18.7% and 1.3%, respectively, compared to 21.2% and 6.7% based on serum cotinine level. Nearly 10.6% and 14.6% of claimed nonsmoker girls and boys were classified as current smokers by serum cotinine level. More than 80% of male smokers started the habit before the age of 20 years and the effect of smoker friends was the most important factor for smoking initiation among smokers. Using self-reported prevalence data for smoking among women or adolescents in special populations like Iranians can give invalid measurements, therefore, data based on biochemical tests are suggested.