CONTEXT: Inter-individual differences in cortisol production and metabolism emerge with age, and may be explained by genetic factors. OBJECTIVE: To estimate the relative contributions of genetic and environmental factors to inter-individual differences in cortisol production and metabolism throughout adolescence. DESIGN: Prospective follow-up study of twins. SETTING: Nationwide register. PARTICIPANTS: 218 mono- and dizygotic twins (N=109 pairs) born between 1995-1996, recruited from the Netherlands Twin Register. Cortisol metabolites were determined in 213, 169 and 160 urine samples at the ages of 9, 12 and 17y, respectively. MAIN OUTCOME MEASURES: The total contribution of genetic factors (broad-sense heritability), and shared and unshared environmental influences to inter-individual differences in cortisol production, and activities of 5α-reductase, 5β-reductase, 11β-hydroxysteroid dehydrogenases and cytochrome P450 3A4. RESULTS: For cortisol production rate at the ages of 9, 12 and 17y, broad-sense heritability was estimated as: 42%, 30% and 0%, respectively and the remainder of the variance was explained by unshared environmental factors. For cortisol metabolism indices, the following heritability was observed: for the A-ring reductases (5α-and 5β-reductases), broad-sense heritability increased with age (to>50%), while for the other indices (renal 11β-HSD2, global 11β-HSD and CYP3A4), the contribution of genetic factors was highest (68%, 18% and 67%, respectively) at age 12y. CONCLUSIONS: The contribution of genetic factors to inter-individual differences in cortisol production decreased between 12 and 17y, indicative of a predominant role of individual circumstances. For cortisol metabolism, distinct patterns of genetic and environmental influences were observed, with heritability that either increased with age or peaked at age 12y.