This article presents the theory and bench validation of access flow measurement by ultrasound velocity dilution. Reversal of dialysis lines creates a zone of mixing in the vascular access, allowing the use of dilution technique for access flow measurement. The method of sound velocity dilution sensor calibration was analyzed. A single sensor system was investigated for blood circulation and isothermal injections. A dual sensor system was investigated with heated hypertonic saline circulation for both body temperature (isothermal) and room temperature (cold) injections. The accuracy of in vitro access flow measurement was studied on a two pump bench model, dialysis line flow was varied from 200 to 400 ml/min, access flow was varied from 300 to 2500 ml/min. For the dual sensor system, dilution access measurements tracked a gravimetrically calibrated in-line flow sensor within 3.25 +/- 0.34% for isothermal injections, and within 5.81 +/- 0.43% for cold injections. Measurements were repeatable within 3% independent of the temperature of the injected saline. For the single sensor system, results tracked the in-line flow sensor within 3.83 +/- 0.79%. These data show that access flow can be accurately measured by sound velocity dilution technique.