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Introduction/Justification: Static renal scintigraphy using 99mTc-DMSA is an accurate method for diagnosing and monitoring renal scars and allows for semi-quantification of relative tubular function (RTF). However, in cases of hydronephrosis, radiopharmaceutical accumulation in the pyelocaliceal system may interfere with RTF quantification. Although 24-hour images are typically requested to address this issue, they can inconvenience patients and disrupt the nuclear medicine service routine. Objectives: This study aimed to assess the impact of additional 24-hour imaging on RTF quantification in patients with hydronephrosis compared to standard 3-hour images. Materials and Methods: A retrospective analysis was conducted on patients who underwent renal scintigraphy with 99mTc-DMSA, focusing on those who received additional 24-hour imaging. Patients were divided into two groups: those aged up to 12 years (Group 1) and those over 12 years old (Group 2). Planar images were acquired 3 hours post-injection of 175 mBq of 99mTc-DMSA for adults and 1.5 MBq/kg for patients weighing up to 40 kg. Additional delayed images were obtained after 24 hours if pyelocaliceal dilation was present. RTF was calculated using both 3-hour and 24-hour images, preferably using semi-automatic regions of interest. The T-Student test was utilized for statistical analysis, considering a difference of ≤ 3% between the two values as not significantly justifying the additional 24-hour image. Results: A total of 1,205 consecutive 99mTc-DMSA scans from February 2019 to December 2023 were evaluated. Group 1 comprised 662 patients, with 62 undergoing additional 24-hour imaging, while Group 2 consisted of 543 patients, with 43 undergoing 24-hour imaging. The mean value of the difference between the 3h and 24h images is 1.95% ± 1.83% and median 2 (0 - 6) for Group 1, and 2.40% ± 2.08% and median 2 (0 - 8) for Group 2. Statistical analysis demonstrated equivalence between RTF quantifications obtained at 3-hour and 24-hour imaging for Group 1 p < 0.0001, 95% confidence interval (1.45 - 2.42). However, for Group 2, quantifications at 3-hour and 24-hour imaging were not necessarily equivalent p = 0.0714, 95% confidence interval (1.76 - 3.05). Conclusion: Additional 24-hour imaging with 99mTc-DMSA in patients under 12 years of age with pyelocaliceal dilation does not appear to impact RTF compared to 3-hour images. However, for older patients, 24-hour imaging is necessary for greater accuracy in RTF determination. Further investigations are warranted to better understand factors influencing RTF calculation, guiding the indication for additional 24-hour imaging.