OBJECTIVE To improve the estimation of 99mTc mercaptoacetyltriglycine clearance in the renal uptake method by optimizing the conditions of renal depth, background, threshold for renal boundary determination, and time interval for integrating renal counts. METHODS Dynamic renal imaging was performed in 232 patients with dual energy window acquisition (main, 140 +/- 14keV; sub, 122.5 3.5keV). For drawing renal regions of interest (ROIs), cut-off methods with 50% and 70% of the highest renal pixel counts were used. For drawing the backgrounds, circumferential and lateral-inferior quadrant peri-renal ROIs were used. For setting the time interval, periods of 1-2, 1-2.5, 1.5-2.5, 1.5-3 and 2-3 min post-injection were used. For determining renal depth, three methods of a theoretical exponential function using scatter fraction, Tønnesen's formula, and linear combination of scatter fraction and Tønnesen's formula were used. The scatter fraction was calculated using the counts in renal ROIs in the two energy windows. Using every combination of these conditions, renal uptake was calculated. As a reference, one-sample clearance was calculated from a blood sample taken at 30 min post-injection following Bubeck's formula. According to the methods for estimating renal depth, three non-linear regression models were derived to convert renal uptake to clearance. Using one-sample clearance and integrated renal counts as dependent and independent variables, data were fitted to the models to determine the necessary constants. The correlations between the model estimated clearances and one-sample clearance were investigated. RESULTS One-sample clearance ranged from 11 to 404 ml x min(-1) per 1.73 m2. More than half of the regression using renal depth determined by the scatter fraction alone failed to converge. Among the successfully converged regressions, all model estimated clearances showed significant correlations (P<0.01) with one-sample clearance. The best correlation was observed in the model using renal depth determined by the combination of scatter fraction and Tønnesen's formulas, renal ROIs by 50% cut-off, lateral-inferior background and time interval of 2-3 min (r=0.898, P<0.001). CONCLUSIONS The renal uptake method for estimating the clearance of mercaptoacetyltriglycine can be improved by the processing conditions proposed here.