Quantitative evaluation of renal function in Tc-99m-DTPA isotope renography is hampered by high background activity. This study compares various background regions of interest (ROIs) to obtain the most accurate estimate of the extra-renal background, taking both total counts (integral value) and change by time (slope) into account. Gamma camera renography was performed in 21 patients with a single kidney. Time-activity histograms obtained from 8 different background ROIs were compared in the time interval corresponding to the uptake phase of the renogram with the histogram obtained from a ROI over the empty kidney area. A significant difference could be demonstrated between the slope values of the histograms of the right and left empty kidney ROIs, but not for the integral values. Backgrounds taken over the heart, liver, between the kidneys, and under the kidneys had significantly different characteristics than the one in the empty kidney area. Only background ROIs surrounding each kidney area gave integral and slope values with close resemblance to those recorded over the empty kidney area. The results emphasize the necessity for an individual background ROI for each kidney and the need to separate extra- and intrarenal background activities before subtraction.