Context: Physiological gynecomastia 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, although this is rarely evident in analyses of serum. Objective: This study aimed to describe the frequency of physiological gynecomastia and to determine possible etiological factors (eg, auxology and serum hormone levels) in a longitudinal setup. 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 6 months during an 8-year follow-up. Median number of examinations was 10 (2–15). Main outcome measurements: Blood samples were analyzed for FSH, LH, testosterone, estradiol, SHBG, inhibin B, anti-Müllerian hormone, IGF-1, and IGF binding protein-3 by immunoassays. Auxological parameters, pubertal development, and the presence of gynecomastia were evaluated at each visit. Results: Fifty-two of 106 boys (49%) developed gynecomastia, of which 10 (19%) presented with intermittent gynecomastia. Boys with physiological gynecomastia reached peak height velocity at a significantly younger age than boys who did not develop gynecomastia (13.5 versus 13.9 years, P = .027), and they had significantly higher serum levels of IGF-1 (P = .000), estradiol (P = .013), free testosterone (P < .001), and FSH (P = .030) during pubertal transition. However, no differences in serum LH or in the estradiol to testosterone ratio were found. Conclusions: Gynecomastia is frequent in pubertal boys. Increased IGF-1 levels and pubertal growth appear to be associated, whereas changes in estrogen to testosterone ratio seem negligible.