Nitric oxide (NO), a selective pulmonary vasodilator, can be delivered via conventional ICU and anesthesia machine ventilators. Anesthesia machines are designed for rebreathing of circulating gases, reducing volatile anesthetic agent quantity used. Current cylinder- and ionizing-based NO delivery technologies use breathing circuit flow to determine NO delivery and do not account for recirculated gases; therefore, they cannot accurately dose NO at FGF below patient minute ventilation (MV). A novel, cassette-based NO delivery system (GENOSYL^® DS, Vero Biotech Inc.) uses measured NO concentration in the breathing circuit as an input to an advanced feedback control algorithm, providing accurate NO delivery regardless of FGF and recirculation of gases. This study evaluated GENOSYL^® DS accuracy with different anesthesia machines, ventilation parameters, FGFs, and volatile anesthetics. GENOSYL^® DS was tested with GE Aisys and Dräger Fabius anesthesia machines to determine NO dose accuracy with FGF < patient MV, and with a Getinge Flow-i anesthesia machine to determine NO dose accuracy when delivering various volatile anesthetic agents. Neonatal and adult mechanical ventilation parameters and circuits were used. GENOSYL® DS maintained accurate NO delivery with all three anesthesia machines, at low FGF with recirculation of gases, and with all volatile anesthetic agents at different concentrations. Measured NO₂ levels remained acceptable at ≤ 1 ppm with set NO dose ≤ 40 ppm. GENOSYL^® DS, with its advanced feedback control algorithm, is the only NO delivery system capable of accurately dosing NO with anesthesia machines with rebreathing ventilation parameters (FGF < MV) regardless of anesthetic agent.