The aim of the study was to examine the prevalence and type of medicines used by high school students in the Czech Republic and to analyze association between use of medicines during last 6 months and self medication with different socio-demographic and lifestyle aspects. A cross-sectional study was conducted in high schools from two regions and three towns in the Czech Republic. The study sample was a student population of nine selected high schools. The study was approved by the local Ethics Committees and school board and was conducted in compliance with the ethical principles of the Declaration of Helsinki. Questionnaires respecting anonymity were distributed to the students during the regular class period. Students answered closed and open questions divided into six sections focussing on their experience with chronic and irregular use of medicines, medicines use during last six months, conditions treated, type and source of used medicines. A modified log-linear Poisson regression model with a robust variance estimator (sandwich) was used for statistical analysis. In total, 979 students, aged 14-21 years (mean age 17 years) participated in the study: 32% were male and 68% female; 19% of students reported regular use of medicines for chronic disease. The use of medicines during the last 6 months was reported by 83% of students; 42% of them reported the use of both prescription and over the counter (OTC) medicines; 22% the use only of OTC, and 31% only prescription medicines. Allergy, asthma, disorders of the thyroid gland, atopic dermatitis, and hypertensive diseases were among the most frequent long-term diseases. The most frequent groups of medicines used for the treatment of long-term diseases were systemic antihistamines, thyroid preparations, and respiratory drugs. Nonsteroidal anti-inflammatory medicines, ibuprofen and paracetamol were the most frequently used medicines during last six months. The study results showed quite extensive use of both prescription and OTC medicines in students of high schools between 14-21 years of age in the Czech Republic. Statistically significant associations were found between use of medicines during last 6 months and type of school, sex and chronic diseases, and between self-medication and sex.