Background: Drug use and abuse may hamper learning capabilities and the development of technical skills in medical students and, therefore, the quality of care offered to patients. The aim of this investigation was to estimate the prevalence of psychoactive drug use among medical students of public universities in Rio de Janeiro, Brazil, and to identify characteristics associated with substance use. Method: This was a cross-sectional investigation designed to include all medical students of four universities. The final sample included 1,054 students. Patterns of licit and illicit drug use (at least once in lifetime drug use, drug use in the last 30 days (LTD) and CAGE) before and during medical school were assessed by a multiple-choice, self-administered anonymous questionnaire. Results: Alcohol abuse was more prevalent among male students from higher income families. Alcohol LTD use was more prevalent among male students with college-educated parents. Tobacco, cannabis and inhalant lifetime use was more prevalent among males and tranquillizer use among females. Tobacco, cannabis and tranquillizer lifetime use was more prevalent among students with divorced or dead parents. Inhalant lifetime use was more prevalent among students from higher income families. Students who had college-educated, divorced or dead parents or evidenced tobacco, cocaine or inhalant lifetime use were more prevalent among cannabis users. Male students from higher income families had higher prevalence of cocaine lifetime use. Conclusion: Substance use in this group of medical students is not widespread compared to rates reported for developed countries. Preventive efforts should focus on alcohol and cannabis use by medical students.