OBJECTIVE To determine effects of the size and location of regions of interest (ROIs) in the renal cortex of unsedated dogs on renal perfusion variables determined by use of contrast-enhanced ultrasonography (CEUS). ANIMALS 12 client-owned adult (1.5 to 2 years old) Labrador Retrievers (8 males and 4 females; mean ± SD body weight, 27 ± 1.6 kg). PROCEDURES Each dog received 2 bolus injections of sulfur hexafluoride during CEUS. Three small oval ROIs (area of each ROI, 0.11 cm(2)) located in a row with a distance of 1 mm between adjacent ROIs and 1 large oval ROI (area, 1 cm(2)) that encompassed the 3 smaller ROIs were manually drawn in the renal cortex. The ROIs were located at a depth of 1.5 to 2.0 cm in the near field of the renal cortex. Software analysis of time-intensity curves within each ROI was used to identify peak enhancement, time to peak enhancement, regional blood flow, and mean transit time. RESULTS The location and size of the ROIs of unsedated dogs did not cause significant differences in the mean values of the renal perfusion variables. CONCLUSIONS AND CLINICAL RELEVANCE The development of CEUS has provided a unique means for visually examining and quantifying tissue perfusion. Results of this study indicated that it was possible to use small or large ROIs during renal CEUS to evaluate renal perfusion in dogs.