OBJECTIVE To evaluate the utility of a method for estimating glomerular filtration rate (GFR) after single IV administration of iohexol. METHODS The plasma clearance of iodine (PCI), taken as the quotient of the administered dose of iodine (300 to 600 mg of l/kg of body weight) divided by the area under the plasma iodine concentration versus time curve determined by 4 methods (PCI1-PCI4). The results for PCI were compared with simultaneously obtained values for the urinary clearance of exogenously administered creatinine (CCr), a widely accepted method for the measurement of GFR in cats and dogs. METHODS Cats and dogs that were renal intact (n = 5 cats; n = 1 dog) or had renal mass reduced by partial nephrectomy (n = 5 cats; n = 7 dogs). RESULTS Values for PCI were closely related (R2 values ranged from 0.947 to 0.992; P < 0.0001 in all cases) to CCr. Despite this close correlation between CCr and PCI, the 95% confidence interval for the difference between PCI3 and CCr included values that exceeded 1.4 ml/min/kg, which represents 50% of the mean value for CCr in renal-intact cats. CONCLUSIONS Determination of PCI provided a reliable estimate of GFR in cats and dogs of this study. However, differences between 1 of the methods (PCI3) and CCr are clinically important, emphasizing the need to use more than simple linear regression analysis and correlation coefficients when attempting to validate new measurement techniques. CONCLUSIONS The determination of PCI provided a reliable estimate of GFR in cats and dogs of this study.