Background/Aim. The most frequent method for the assessment of glomerular filtration rate GFR) in clinical practice is clearance of creatinine, clearance of chromium-51 radiolabelled ethylene diamine tetraacetic acid (51Cr-EDTA) and clearance of technetium-99m radiolabelled diethylene triamine pentaacetic acid (99mTc-DTPA). The Gates method for glomerular filtration rate assessment is based on distribution of 99mTc-DTPA in the kidney 2-3 minute after its applying. Calculation of GFR is corrected for the background and depth of the kidney and finally expressed as a percentage of the net injected counts. This value of GFR highly correlates with biexponential model as the most accurate method for the assessment of GFR. The aim of this study was to determine the influence of different background sites on GFR rate assessment using the Gates method. Methods. We analysed 50 patients who were divided into two groups: the group of healthy subjects (21 subjects, mean age 47.38 yrs) and the group of subjects with unilateral kidney damage (29 subjects, mean age 39.79 yrs). Three different background activity regions were chosen: a region drawn below the lower pole of each kidney, region drawn by the lateral side of each kidney and region drawn around the whole kidney. Results. Gromerular filtration rate calculated by the use of a region under the lower pole is statistically significantly higher than GFR calculated using a region by the lateral side and around the whole kidney (p < 0.0001). Glomerular filtration rate calculated using the region by the lateral side and region around the whole kidney did not show statistically significant difference (p > 0.05). Conclusion. The selection of background activity region has a significant influence on GFR rate measured by the Gates method. It is recommended to use only one method for choosing the region of background activity.