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Although an outstanding characteristic of the adrenocortical function of children and adults is its circadian rhythm, little information is available about the age of appearance of such rhythm in infancy. The main obstacle has been the ethical difficulty in obtaining serial blood samples from healthy infants. We monitored the development of cortisol daily rhythm using non-invasive salivary cortisol determination.A longitudinal study of a group of 9 healthy infants was performed. Salivary samples were obtained in the morning, afternoon and night at 2, 4, 8, 12, 16, 20 and 24 post-natal weeks from all infants.Cortisol was determined by RIA in 25-μl salivary samples. Two techniques based on assay coefficients of variation were employed to characterize a normal circadian pattern of cortisol.Five infants (55%) established and maintained their cortisol rhythm as early as at 2, 4 and 8 weeks of age. In the remaining 4 infants the age of appearance was 12 and 20 weeks. This rhythm emerged in the group as a whole at a mean age of 8 weeks.Our data suggest that, in most normal infants, the development of hypothalamic-pituitary-adrenal axis circadian maturation may occur at a much earlier age than previously described.