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Dynamic renal scintigraphy remains the gold standard for assessing differential renal function (DRF). Recently, technetium-99m-ethylenedicysteine (99mTc-EC) was shown to be valuable and had similar quality images as technetium-99m-mercaptoacetyltriglycine (99mTc-MAG3). However, its reproducibility has never been confirmed. The aim of this study was to perform the first evaluation of 99mTc-EC reproducibility for assessing DRF in children who were referred for hydronephrosis or urinary tract dilatation.A total of 109 patients from three French nuclear medicine departments prospectively underwent dynamic renal scintigraphy with 99mTc-EC. DRF reproducibility was assessed by different pairs of raters using a multilevel design that integrated local and centralized predefined procedures.Both local and centralized procedures yielded near-excellent inter-rater agreements, with all of the intraclass correlation coefficient values over 0.998. Bland–Altman plots showed a systematic bias of less than 1%, with the corresponding limits of agreements not exceeding the 5% threshold cut-off value that corresponds to the clinical definition of acceptable limits for this purpose. Intrarater agreements were also good to excellent.This prospective multicentre study showed that 99mTc-EC is highly reproducible for assessing DRF in a standard paediatric population, thus validating its use as an alternative to 99mTc-MAG3 in this setting.