Purpose: To determine appropriate sampling frequency and time of multiple blood sampling dual exponential method with 99mTc-DTPA for calculating glomerular filtration rate (GFR). Materials & Methods: Thirty four patients were included in this study. Three mCi of 99mTc-DTPA was intravenously injected and blood sampling at 9 different times, 5ml each, were done. Using the radioactivity of serum, measured by gamma counter, the GFR was calculated using dual exponential method and corrected with the body surface area. Using spontaneously chosen 2 data points of serum radioactivity, 15 collections of 2-sample GFR were calculated. And 10 collections of 3-sample GFR and 12 collections of 4-sample GFR were also calculated. Using the 9-sample GFR as a reference value, degree of agreement was analyzed with Kendall's τcorrelation coefficients, mean difference and standard deviation. Results: Although some of the 2-sample GFR showed high correlation coefficient, over or underestimation had evolved as the renal function change. The 10-120-240 min 3-sample GFR showed a high correlation coefficient (τ=0.93), minimal difference (Mean±SD=-1.784±3.972), and no over or underestimation as the renal function changed. The 4-sample GFR showed no better accuracy than the 3-sample GFR. Conclusions: In the wide spectrum of renal function, the 10-120-240 min 3-sample GFR could be the best choice for estimating the patients' renal function. (Nucl Med Mol Imaging 2006;40(1):33-39)