Background: 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 future reproductive function. Aim: The objective of this study was to investigate the postnatal activation of the hypothalamic-pituitary-gonadal axis in female very-low-birth-weight infants. Study design: We performed serial measurements of gonadotropin and estradiol levels in urine samples of female very-low-birth-weight infants collected at 1 and 4. weeks postnatal age, at 32. weeks postmenstrual age, at expected date of delivery and at the corrected age of three and six months. Subjects: Twenty-two very-low-birth-weight infants (gestational age 25.4-30.1. weeks), participating in the Neonatal Insulin Replacement Therapy in Europe trial, were included in this study. Outcome measures: Gonadotropin and estradiol levels were measured in serial urine samples. Results: Longitudinal analysis shows that after birth FSH and LH levels increase until 32. weeks postmenstrual age (4. weeks postnatal age) and then decrease until 3. months corrected age (26. weeks postnatal age). Estradiol levels decrease from 28. weeks postmenstrual age (1. week postnatal age) until 6. months corrected age (39. weeks postnatal age). Conclusions: Serial urine sampling for measurement of gonadotropin and estradiol levels provides an accurate description of the postnatal activation of the hypothalamic-pituitary-gonadal axis in very-low-birth-weight girls. Levels of FSH and LH peak at a mean postmenstrual age of 32. weeks (postnatal age of 4. weeks) whereas estradiol levels are highest shortly after birth. © 2012 Elsevier Ltd.