To find the time of the serum gonadotropin peak after depot leuprolide injection in children and to show that depot leuprolide therapy can be monitored by measuring serum luteinizing hormone (LH) immediately after injections. We measured concentrations of leuprolide, LH, and follicle-stimulating hormone (FSH) at multiple time points before and after the first dose of depot leuprolide in 14 pubertal children beginning therapy. Gonadotropins and sex steroids were measured again after the fourth dose. Serum leuprolide, LH, and FSH levels rose rapidly after initial injection, reaching sustained elevations at 30 to 120 minutes. The median LH level increased from 2.1 mIU/mL at baseline to a peak of 27.5 mIU/mL at 45 minutes, and FSH increased from 5.2 to 16.5 mIU/mL. After 3 months on therapy, median serum LH after depot leuprolide injection was only 0.83 mIU/mL, similar to levels observed after intravenous or subcutaneous gonadotropin-releasing hormone stimulation in comparable subjects on depot leuprolide. Our pharmacokinetic data demonstrate that free leuprolide present in a depot leuprolide injection is equivalent to gonadotropin-releasing hormone in stimulating a rapid rise in serum gonadotropin concentrations. We propose that a single serum sample for LH obtained 30 to 60 minutes after depot leuprolide injection in children provides a convenient and accurate assessment of treatment efficacy.