Extracellular fluid volume (ECV) is studied infrequently. The zero-time distribution volume (Vd) generated in the slope-intercept technique for measuring the glomerular filtration rate (GFR) substantially overestimates ECV. The aim was to validate a new technique for measuring ECV from the slope-intercept approach. GFR and ECV were measured using Cr-51-EDTA and iohexol injected into opposite arms in 51 patients undergoing routine measurement of GFR and on 48 occasions in 20 healthy volunteers. Blood samples were obtained bilaterally 20, 40, 60, 120, 180 and 240 min post-injection and assayed for indicator injected contralaterally. Reference ECV (ECV6) was calculated from all six samples as the product of indicator transit time and multi-sample GFR. GFR/ECV was calculated as the rate constant of the exponential fitted to the last three samples (GFR/ECV3). Slope-intercept GFR was calculated from the last three samples using the slope-intercept technique (GFR3). ECV (ECV3) was calculated by dividing GFR3 by GFR/ECV3, having corrected both for their one-compartment assumptions. ECV6_EDTA correlated closely with ECV3_EDTA (ECV3_EDTA = 1.01.ECV6_EDTA–0.5 L; r = 0.97; n = 99), but less closely with Vd (Vd = 1.17.ECV6_EDTA + 0.7 L; r = 0.86). ECV6_iohexol correlated slightly better with ECV6 _EDTA (ECV6_EDTA = 0.81.ECV6_iohexol + 3.3 L; r = 0.86) than with ECV3_EDTA (ECV3_EDTA = 0.83.ECV6_iohexol + 2.9 L; r = 0.84) and had slightly narrower 95% limits of agreement (−3.82 and 2.82 L versus −3.90 to 3.43 L). In conclusion, ECV can be measured from three samples almost as accurately as ECV from multiple samples.