Thyroid-to-salivary ratio and percent dose uptake are the most widely recognized scintigraphic measurements. Recently, the thyroid-to-background ratio has been proposed as an alternate method. However, this method has not been validated. The purpose of this observational, cross-sectional, prospective study was to determine the location of a background region of interest (ROI) that is most reflective of blood pool activity. We also hypothesized that the thyroid-to-background ratio using this background ROI would be a better predictor of thyroid function. Fifty-six cats presented to the Virginia-Maryland College of Veterinary Medicine seeking radioiodine therapy for hyperthyroidism were enrolled in this cross-sectional study to evaluating thyroid-to-background ratio. A blood sample for measuring plasma radioactivity was collected at the time of scintigraphy. The plasma radioactivity was compared to the background ROIs in eight anatomic regions. Scintigraphic measures of thyroid-to-background and thyroid-to-salivary ratios, and percent dose were then compared to serum T4. The heart ROI was most closely correlated with plasma pertechnetate activity (r= 0.70). Percent dose uptake was most closely correlated with serum T4 (r= 0.74), followed by thyroid-to-salivary ratio (r= 0.66) and thyroid-to-background ratio using the heart ROI (r= 0.59). Thyroid-to-background ratio using the heart background ROI is a good predictor T4 but percent dose uptake and thyroid-to-salivary ratio proved to be better predictors of T4 than any of the thyroid-to-background ratios.