Determination of glomerular filtration rate (GFR) with 99mTc-labeled diethylenetriaminepentaacetic acid (99mTc-DTPA) is a validated method to assess renal function, but there are no well-established pediatric reference ranges for GFR. The aim of this study was to establish reference GFRs in children and young adults. Methods: GFR was calculated for 235 reference subjects (median age, 4.86 y; range, 20 d to 24.8 y) using 99mTc-DTPA at Boston Children's Hospital between 2000 and 2020. This included 220 patients for prechemotherapy evaluation and 15 potential kidney donors. GFR was determined using a single-compartment model after a single intravenous injection technique and serial blood sampling at 2, 3, and 4 h. Data were analyzed by age, corrected for body surface area, and normalized to a body surface area of 1.73 m2 The 95% reference ranges were calculated as mean ± 1.96 SD. Results: GFR increased with age, likely reflecting increased functional renal mass. Lower normalized GFR in children younger than 1 y may reflect renal immaturity. Conclusion: The use of a single injection of 99mTc-DTPA revealed that GFR increases with age, likely reflecting increased renal mass. Further, in patients younger than 1 y, GFR rapidly increases initially, with a subsequent more gradual increase, reaching the adult level by approximately 2 y of age. Between 2 and 10 y of age, the GFR slightly exceeds the adult level, stabilizing at a slightly lower adult level by 10 y of age.