Hemodynamic stability during hemodialysis depends largely on plasma volume (PV) preservation during ultrafiltration (UF). Current estimates of blood volume (BV) are indirect or involve the use of radioactive tracers, which does not allow repeated measurements during hemodialysis. Indocyanine green was used to measure PV during hemodialysis. After an initial pilot phase (phase I), PV values were determined before dialysis, repeatedly during isovolemic hemodialysis (phase II), and during stepwise UF (phase III). Absolute BV values were calculated from PV and hematocrit values. Patients were monitored for extracellular fluid volume (bioimpedance monitoring) and relative BV changes (ultrasonic monitoring). Phase I demonstrated dye stability in plasma, peak absorbance at 805 nm, and a short half-life (4.53 +/- 1.5 min). Ten milligrams of dye (2.5 mg/ml) were injected for each PV measurement. Eight plasma samples were obtained beginning 3 min after injection, at 1-min intervals, for assessment of decay characteristics. The isovolemic hemodialysis PV measurements demonstrated excellent reproducibility (r(2) = 0.98; method SD, 356 ml; mean coefficient of variation, 4.07%) and a difference of only 149 +/- 341 ml (mean +/- SD), compared with predialysis PV values (Bland-Altman method). PV values at the beginning of dialysis were significantly correlated with body surface area (r(2) = 0.82, P < 0.001) and extracellular fluid estimates (r(2) = 0.73, P < 0.001). BV prediction formulae significantly underestimated absolute BV at the start of dialysis (P < 0.0001). The findings demonstrate that this method can be used for repeated PV determinations during hemodialysis, with excellent reproducibility. It is a potential tool for further research on hemodynamic stability during UF.