Background/Aims: The postnatal activation of the hypothalamic-pituitary-gonadal axis is more exaggerated in preterm than in full-term-born infants, and may be important for reproductive function. Our objective was to investigate this activation of the hypothalamic-pituitary-gonadal axis in male very-low-birthweight (VLBW) infants. Methods: Twenty-one VLBW boys (gestational age 26.0-30.0 weeks), participating in the NIRTURE trial, were included. Gonadotropin and testosterone levels were measured in serial urine samples collected at 1 and 4 weeks' postnatal age, at 32 weeks' postmenstrual age, at expected date of delivery and at the corrected age of 3 and 6 months. Results: Longitudinal analysis shows that after birth LH and FSH levels peak at a mean postnatal age of 1-4 weeks (mean postmenstrual age of 30-32 weeks) and decrease until 38 weeks' postnatal age (corrected age of 6 months). Testosterone levels decrease with increasing age, and this decrease is faster in infants receiving early insulin therapy. Conclusions: Serial urine sampling for measurement of gonadotropin and testosterone levels provides accurate information about the postnatal activation of the hypothalamic-pituitary-gonadal axis in VLBW boys. FSH and LH levels peak at 1-4 weeks of age. Insulin treatment causes faster decrease in testosterone levels. Copyright © 2012 S. Karger AG, Basel.