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Background: The normal development of cortisol regulation during childhood is thought to be influenced by a complex interplay between environmental and genetic factors. Method: The aim of this study was to estimate genetic and environmental influences on basal cortisol levels in a sample of 151 twin pairs aged 9–16 years. Salivary cortisol was collected on two consecutive days when the children attended school — immediately after awakening, 30 min post-awakening and at bedtime. Results: Heritability was highest (60%) for cortisol levels about 30 min after awakening. For samples taken immediately at awakening heritability was less pronounced (28%) and in the evening low (8%). Conclusion: The limited genetic influence on evening levels, moderate on cortisol at awakening and high on awakening response, might imply two genetic regulation patterns, one specifically for awakening response and one for the circadian rhythm proper. These findings could explain divergent results in previous studies and highlight the importance of taking the circadian rhythm into account in studies of cortisol levels in children.