Chronic biological stress may adversely affect adolescents' physical and mental health, but insight in the personal and environmental factors that determine chronic stress is limited. We measured 3-month cumulative hair cortisol concentration (HCC) in 419 adolescents, participating in the Flemish Environment and Health Study. Adolescents' health and lifestyle characteristics, household and neighborhood socio-economic status as well as neighborhood urbanicity were assessed as potential determinants of HCC, using multiple linear regression models. We additionally explored heterogeneity of our results by sex. HCC were significantly higher in boys from densely populated neighborhoods, the association was not significant in girls. Accordingly, boys living outside cities had significantly lower HCC than boys, living in cities. HCC was significantly lower in adolescents with an optimal vitality, a measure of a positive mental health status. In adolescent girls, menarcheal status (pre-/postmenarche) was a significant determinant of HCC. Our findings are the first to suggest that residential urbanicity may have an impact on chronic biological stress in a general population of adolescent boys.