Background Blood volume quantification is essential for haemodynamic evaluation guiding fluid management in anaesthesia and intensive care practice. Ultrasound contrast agent (UCA)-dilution measured by contrast enhanced ultrasound (CEUS) can provide the UCA mean transit time (MTT) between the right and left heart, enabling the assessment of the intrathoracic blood volume (ITBVUCA). The purpose of the present study was to investigate the agreement between UCA-dilution using CEUS and transpulmonary thermodilution (TPTD) in vitro and in vivo. Methods In an in vitro setup, with variable flows and volumes, we injected a double indicator, ice-cold saline with SonoVue®, and performed volume measurements using transesophageal echo and thermodilution by PiCCO®. In a pilot study, we assigned 17 patients undergoing elective cardiac surgery for pulmonary blood volume (PBV) measurement using TPTD by PiCCO® and ITBV by UCA-dilution. Correlation coefficients and Bland-Altman analysis were performed for all volume measurements. Results In vitro, 73 experimental MTT's were obtained using PiCCO® and UCA-dilution. The volumes by PiCCO® and UCA-dilution correlated with true volumes; rs=0.96 (95% CI, 0.93–0.97; P<0.0001) and rs=0.97 (95% CI, 0.95–0.98; P<0.0001), respectively. The bias of PBV by PiCCO® and ITBVUCA were −380 ml and −42 ml, respectively. In 16 patients, 86 measurements were performed. The correlation between PBV by PiCCO® and ITBVUCA was rs=0.69 (95% CI 0.55–0.79; P<0.0001). Bland-Altman analysis revealed a bias of −323 ml. Conclusions ITBV assessment with CEUS seems a promising technique for blood volume measurement, which is minimally-invasive and bedside applicable.