Determinations of different methods of measurement of uptake of 99mTc-DTPA using scintigraphy of glomerular filtration rate (GFR) were made from 29 studies on 10 healthy beagle dogs. GFR was measured by calculating the percentage dose uptake (integral method) and rate of uptake (slope method) of 99mTc-DTPA using manual kidney regions of interest (ROI) and automatic kidney and background ROIs at different time periods of the uptake phase. These results were compared using linear regression analysis to the GFR obtained from 99mTc-DTPA plasma clearance using multiple blood samples. The best correlation coefficient between percentage DTPA uptake and GFR by DTPA clearance (r = 0.84, P < 0.001) was derived from time intervals between 30s–120s with a perirenal background ROI at 1 or 2 pixels out from the kidney ROI using automatic kidney ROI at 20% threshold. With the slope method, the best correlation coefficient (r = 0.85, P < 0.001) was obtained from time intervals between 30s-peak with the background ROI at 2 pixels out from the kidney ROI using automatic ROI at 35% threshold. The offset was higher, and the correlation varied more with different ROIs and the method was unreliable at time intervals extending beyond the peak radioactivity. Manual kidney ROIs with automatic background ROIs had slightly lower correlations. With DTPA renography both integral and slope uptake method with automatic kidney and background ROIs are accurate methods to estimate the GFR, but that the integral method is much more stable to variations in ROI size and the duration of the uptake phase of the renogram.