Objective: To evaluate the feasibility of semen cryopreservation in pubertal boys before they receive gonadotoxic therapy and to identify which pretreatment parameters might predict successful cryopreservation. Design: Retrospective data analysis. Setting: Tertiary fertility center, academic children's hospital. Patient(s): Between 1995 and 2005, 80 boys (median age 16.6 years, range 13.7-18.9 years) consulted the outpatient clinic of andrology for semen cryopreservation before a potentially gonadotoxic treatment. Intervention(s): We assessed the pretreatment semen parameters, hormone levels, and patients' characteristics. Main Outcome Measure(s): Measurement of the number of adolescents able to cryopreserve semen. Result(s): Thirteen boys were unable to produce semen by masturbation. In 53 boys semen quality was adequate for cryopreservation. In 14 patients semen analysis did not show motile spermatozoa, and therefore semen cryopreservation could not be performed. Although inhibin B showed a strong correlation with sperm count, no significant difference was found in serum T, inhibin B, LH, and FSH levels in the patients with or without successful sperm yield. Moreover, median age was not different between patients with and without a successful sperm yield. Conclusion(s): Semen cryopreservation in boys is a feasible method to preserve spermatozoa before gonadotoxic therapy is started and should be offered to all pubertal boys despite their young age. Serum hormone levels do not predict sperm yield.