Normal children and adults show diurnal variation of plasma cortisol levels reaching peak values around 08.00 h and lower values around 24.00 h. Despite numerous studies on diurnal variation of plasma cortisol levels in children, the age of appearance of a circadian rhythm has not been definitely established. The purpose of this study was to investigate the development of cortisol circadian rhythm in infancy. In seventy healthy, full-term infants, less than six months old, plasma cortisol was measured at 10.30 h and 22.30 h. The mean +/- SEM values of daytime plasma cortisol at one to four weeks of life were 159 +/- 41, 116 +/- 43, 240 +/- 54 and 456 +/- 95 nmol/l and the night-time values were 129 +/- 40, 99 +/- 44, 131 +/- 78 and 430 +/- 105 nmol/l, respectively. No statistically significant differences were found between daytime and night-time cortisol values during the first four weeks of life. In contrast, the daytime values of plasma cortisol were 295 +/- 62 at the age of 2-3 months, 211 +/- 43 at 4-5 months and 291 +/- 31 nmol/l at 6 months of life, and night-time values were 166 +/- 52, 119 +/- 35 and 109 +/- 21 nmol/l, respectively, which were statistically significant (p < 0.05). These data clearly indicate that cortisol circadian rhythm starts between the second and third month of life.