Tc-99m-L,L-ethylene, L, dicysteine (EC) clearance shows strong correlation with orthoiodohippurate clearance, and it is possible to estimate effective renal plasma flow from Tc-99m-EC clearance. In routine clinical studies, it is practical to use the one or two plasma sample method instead of multiple plasma samples for clearance determination. A single-sample technique was developed for Tc-99m-EC, and a regression formula was generated. A prospective study tested the validity of this regression formula. Methods: The study population was composed of 26 patients with a wide range of renal function. Multiple plasma sample Tc-99m-EC clearances were calculated from all patients using the open two-compartment model, Single plasma sample clearances were also determined from the 54-min plasma sample using the regression formula published previously. Results: The multiple-sample plasma clearance of Tc-99m-EC ranged from 46 to 668 mL/min with a mean of 300.76 +/- 150.73 mL/min. The clearances obtained from the 54-min plasma sample ranged from 49 to 699 mL/min, with a mean of 297.39 +/- 152.23 mL/min, There was an excellent correlation between the clearances obtained by the two techniques (r = 0.99, slope = 0.9911). The standard error of estimation was found to be 25.9 mL/min, Conclusion: This study suggests that Tc-99m-EC clearance can be estimated from 54-min plasma samples with an acceptable error of estimation for most routine clinical studies.