Tc-99m DMSA renal cortical scan is a reference method for determining relative renal function (RRF). Tc-99m MAG-3 is also recommended for the estimation of RRF, particularly in young children. The aims of this study were to compare MAG-3 and DMSA RRF estimations and to assess the reproducibility of these estimations in children with unilateral hydronephrosis. We reviewed MAG-3 and DMSA scans of 19 children with unilateral hydronephrosis and a normal contralateral unit. All were imaged with 2 agents within 3 months. MAG-3 RRF was calculated using 2 different time intervals (1-2 and 2-3 min) and perirenal C-type region of background activity. No background correction method was used in the calculation of DMSA RRF. All data were processed twice by two independent operators. In the repeated estimates of MAG-3 RRF, the maximum mean intra-observer and inter-observer differences were 0.9% (SD 1.7%) and 1.4% (SD 3.3%), respectively. Intra-method analysis showed statistically significant agreement between repeated measures of RRF for two agents as well as for two operators (intra-class cross-correlation coefficients of early and later phase of MAG-3 and DMSA RRF values were 0.979, 0.993 and 0.996 for operator 1, and 0.986, 0.973 and 0.995 for operator 2, respectively; p < 0.001). There were statistically significant correlations between the MAG-3 and DMSA RRF estimations (r = 0.763/0.766 at 1-2 min, 0.835/0.825 at 2-3 min for the repeated measures of RRF for operator 1, and 0.812/0.793 and 0.83/0.89 for operator 2, respectively; p < 0.01, Pearson). Each scan classified as normal or abnormal according to RRF values inter-method analysis showed statistically significant agreement between MAG-3 and DMSA RRF estimations (Kappa statistics). However, there was a slight disagreement between the methods especially in infants and cases with higher grade of hydronephrosis, when 5% difference in estimations and supranormal estimates of RRF were taken into consideration. Although DMSA and MAG-3 RRF estimations demonstrated a statistically significant correlation with good reproducibility in children with unilateral hydronephrosis, overall the finding of surprising estimates of kidney function with MAG-3 calls attention to the existence of some problems especially in infants and in those with higher grades of hydronephrotic kidneys.