Discordance between effective renal plasma flow (ERPF) measurements from radionuclide techniques that use single versus multiple plasma samples was investigated. In particular, the authors determined whether effects of variations in distribution volume (Vd) of iodine-131 iodohippurate on measurement of ERPF could be ignored, an assumption implicit in the single-sample technique. The influence of Vd on ERPF was found to be significant, a factor indicating an important and previously unappreciated source of error in the single-sample technique. Therefore, a new two-compartment, two-plasma-sample technique was developed on the basis of the observations that while variations in Vd occur from patient to patient, the relationship between intravascular and extravascular components of Vd and the rate of iodohippurate exchange between the components are stable throughout a wide range of physiologic and pathologic conditions. The new technique was applied in a series of 30 studies in 19 patients. Results were compared with those achieved with the reference, single-sample, and slope-intercept techniques. The new two-compartment, two-sample technique yielded estimates of ERPF that more closely agreed with the reference multiple-sample method than either the single-sample or slope-intercept techniques.