Recent clinical studies have questioned whether there is a qualitative change in the circulating isoforms of LH and FSH after stimulation by GnRH. The present study investigated the median charge of serum gonadotropin isoforms before and after an exogenous challenge of 100 micrograms GnRH in 10 girls and 10 boys undergoing pubertal development. All of the children had basal serum levels of LH and FSH and responses 30 and 60 min after GnRH treatment that were considered normal for puberty. LH and FSH in serum and eluates after electrophoresis in 0.10% agarose suspension were measured with sandwich fluoroimmunoassays. The increases in serum gonadotropin concentrations were generally similar for both sexes, but girls had significantly (P < 0.05) higher LH levels at 30 and 60 min than boys and a larger (P < 0.05) relative increase in serum FSH levels after GnRH treatment. In terms of the median charge of serum isoforms, the girls had significantly less negative (i.e. more basic) isoforms of LH (P < 0.05) and FSH (P < 0.001) than the boys. There was a change to more basic isoforms of both LH and FSH in all children 30 min after GnRH administration. For LH, the charge had returned to pretreatment values by 90 min after GnRH, but for FSH, the charge was still significantly (P < 0.05) more basic at this time (n = 4/sex). When the LH isoforms were more acidic before GnRH treatment, the change in median charge was larger than when the isoforms were more basic beforehand. A similar relationship was not found for FSH. Conversely, there was for FSH, but not for LH, a significant (P < 0.001) relationship between the relative increase in serum concentrations and the change in charge of the isoforms 60 min after GnRH treatment. These findings show that the circulating forms of LH and FSH become more basic after an exogenous challenge of GnRH in children undergoing pubertal development and suggest that the differences in the responses of LH and FSH isoforms may be due to differing degrees of selective secretion and/or survival.