Background: Few studies have investigated physiological stress (re)activity in relation to substance use, especially in adolescents. Using substances is one way to stimulate physiological arousal, therefore inherent hypo-arousal may be associated with substance use in adolescents. The purpose of this study was to examine the relation of autonomic nervous system (ANS) activity with alcohol and tobacco use in adolescents. Methods: ANS activity and perceived stress during a social stress procedure were examined in relation to substance use. 275 Dutch adolescents from a general population study provided complete data. Heart rate was recorded continuously during a pre-task rest period, a stressful task period and a post-task recovery period. Alcohol and tobacco use were self-reported. Results: Adolescents who consumed a medium and high number of alcoholic drinks per week (more than two) exhibited lower heart rates during the entire stress procedure as compared to those who consumed a low number of alcoholic drinks. Adolescents who smoked every day portrayed blunted heart rate reactivity to stress as compared to adolescents who smoked less frequently or not at all. Perceived stress was not related to alcohol or tobacco use. Conclusions: Overall lower heart rate in adolescents who drank more and blunted heart rate reactivity to stress in those who used tobacco every day may indicate inherent hypo-arousal of the ANS system in those vulnerable to use substances more often. These adolescents may actively seek out substances in order to achieve a more normalized state of arousal. © 2012 Elsevier Ireland Ltd.