To determine the extent to which video games include alcohol and tobacco content and assess the association between playing them and alcohol and smoking behaviors in adolescent players in Great Britain. Assessment of substance in the 32 UK bestselling video games of 2012/2013; online survey of adolescent playing of 17 games with substance content; and content analysis of the five most popular games. A total of 1,094 adolescents aged 11–17 years were included as participants. Reported presence of substance content in the 32 games; estimated numbers of adolescents who had played games; self-reported substance use; semiquantitative measures of substance content by interval coding of video game cut scenes. Nonofficial sources reported substance content in 17 (44 percent) games but none was reported by the official Pan European Game Information (PEGI) system. Adolescents who had played at least one game were significantly more likely ever to have tried smoking (adjusted odds ratio [OR] 2.70, 95 percent confidence interval [CI] 1.75–4.17) or consumed alcohol (adjusted OR 2.35, 95 percent CI 1.70–3.23). In the five most popular game episodes of alcohol actual use, implied use and paraphernalia occurred in 31 (14 percent), 81 (37 percent), and 41 (19 percent) intervals, respectively. Tobacco actual use, implied use, and paraphernalia occurred in 32 (15 percent), 27 (12 percent), and 53 (24 percent) intervals, respectively. Alcohol and tobacco content is common in the most popular video games but not reported by the official PEGI system. Content analysis identified substantial substance content in a sample of those games. Adolescents who play these video games are more likely to have experimented with tobacco and alcohol.