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Kidney transplant obstruction (KTO) following renal transplantation remains an important reversible cause of allograft dysfunction, requiring prompt diagnosis to prevent long-term graft damage. Although ultrasound can accurately diagnose renal transplant hydronephrosis, it cannot be used to assess its functional significance. We prospectively assessed the utility of technetium-99m mercaptoacetyltriglycine (Tc99m MAG3) diuretic renography for the diagnosis of allograft KTO, using standard visual and quantitative parameters, as well as calculated renal output efficiency (OE), which has been postulated to improve diagnostic yield. From a cohort of 45 renal transplant patients, two subgroups were formed. The first group of transplant recipients (n = 21) with stable function and no obstruction was used to derive normal values for Tc99m MAG3 scans. A second group of transplant recipients with acute renal dysfunction in whom KTO was clinically suspected was used to test the diagnostic utility of these derived values (n = 43 scans). KTO was diagnosed independently of the MAG3 scans by a fall in the serum creatinine in response to renal pelvis urinary drainage. OE in 12 renal allografts with KTO was significantly reduced compared with 31 Tc99m MAG3 scans without KTO (59.6 ± 18.9 vs. 81.6 ± 5.4%, p < 0.001). In KTO, the mean time of isotope appearance in the bladder (time to bladder [TTB]) was extended compared with unobstructed allografts (7.9 ± 4.1 vs. 3.6 ± 1.5 min, p < 0.001). Measurement of OE significantly improved the accuracy of diuretic MAG3 renography in the diagnosis of renal allograft KTO, especially when supplemented by the TTB, parenchymal transit time and shape of the renogram curve. Ureteric obstruction of the kidney transplant can be diagnosed with an OE reduced to < 75% (sensitivity 92%, specificity 87%) and confirmed by isotope hold-up in the pelvicalyceal system. A normal or slowly declining renogram curve effectively excluded KTO (sensitivity of 96%, negative predictive value of 84%). A parenchymal transit time of > 5 min and a TTB of > 7 min both yielded a sensitivity of 92% and a specificity of 81%. In conclusion, MAG3 renography is a clinically useful investigation for the diagnosis of KTO.