CYP2A6 influences smoking uptake in adolescence. Genetic variation in the CHRNA5–CHRNA3 region influences smoking behaviour in adults. However, their combined effects on smoking in adolescence have not been tested to date. We present data on 1450 adolescents from the Ten Towns Heart Health Study (TTHHS) extensively phenotyped for smoking-related traits during adolescence. Single nucleotide polymorphisms from CHRNA5 and CHRNA3 (previously associated with smoking), were typed in our study population, previously genotyped for CYP2A6. Association analyses between each genotype and both smoking status and behavioural markers of smoking were performed. rs16969968 in CHRNA5 was associated both at 13–15 years and 18 years with current smoking amongst adolescents who had tried smoking (OR = 1.82, CI = 1.10–3.01, p = 0.02 at age 13–15; OR = 2.39, CI = 1.37–4.17, p = 0.002 at age 18). No association was found for rs578776 in CHRNA3. The effects of CHRNA5 and CYP2A6 genotypes in TTHHS appeared to be independent, with each approximately doubling the odds of being a regular smoker by age 18 years. CYP2A6 genotype insufficiency increases adolescent likelihood of being a regular smoker but increases later life quitting likelihood and reduces average consumption. In contrast, CHRNA5 genotype, acting recessively, affects smoking similarly in adolescents and older adults. These contrasting actions, in digenic combination, illustrate behavioural genetic complexity.