Context: Physiological gynaecomastia is common and affects a large proportion of otherwise healthy adolescent boys. It is thought to be caused by an imbalance between estrogen and testosterone, though this is rarely evident in analyses of serum. Objective: This study aimed to describe the frequency of physiological gynaecomastia, and to determine possible etiological factors (e.g. auxology and serum hormone levels) in a longitudinal set-up. Design, Settings and Participants: A prospective cohort study of 106 healthy Danish boys (5.8–16.4 years) participated in the longitudinal part of “the COPENHAGEN Puberty Study”. The boys were examined every six months during an eight year follow-up. Median number of examinations was 10 (2–15). Main outcome measurements: Blood samples and analysed for FSH, LH, testosterone, estradiol, SHBG, inhibin B, AMH, IGF-I and IGFBP-3 by immunoassays. Auxological parameters, pubertal development and the presence of gynaecomastia were evaluated at each visit. Results: 52 of 106 boys (49 developed gynaecomastia of which 10 (19 presented with intermittent gynaecomastia. Boys with physiological gynaecomastia reached peak height velocity at a significantly younger age than boys who did not develop gynaecomastia (13.5 vs 13.9 years, p = 0.027), and they had significantly higher serum levels of IGF-I (p = 0.000), estradiol (p = 0.013), free-testosterone (p <0.001) and FSH (p = 0.030) during pubertal transition. However, no differences in serum LH or in the estradiol to testosterone ratio were found. Conclusions: Gynaecomastia is frequent in pubertal boys. Increased IGF-I levels and pubertal growth appear to be associated, whereas changes in estrogen to testosterone ratio seem negligible.