Background: Distribution of ventilation and pulmonary perfusion are the major determinants of pulmonary gas exchange. To study and compare strategies of mechanical ventilation in respiratory research accurate and high-resolution methods are needed to derive distribution of ventilation and perfusion with minimal additional intervention or radiation allowing repeated measurements. Dual-energy computed tomography (DECT) is an imaging technique allowing for the derivation of regional pulmonary perfused blood volume, as a surrogate for pulmonary perfusion (PP_DECT). Here accuracy of PP_DECT is evaluated in comparison to pulmonary blood flow measured with fluorescence-labeled microspheres (PP_FLM). Its feasibility of repeated measurements is evaluated. Methods: Agreement between PP_FLM and PP_DECT was assessed by regression as well as Bland–Altman analysis in three anesthetized pigs using DECT and fluorescence labelled microspheres, respectively. Measurements were performed in two-lung and, after right sided thoracotomy, at one-lung ventilation with inhaled nitric oxide. PP_FLM and PP_DECT were assessed in three different regions of interest (ROI): the right (non-ventilated) and left (ventilated) upper and lower lung, yielding a total of 45 paired measurements over four hours. Persistent iodine accumulation was assessed by additional DECT scans before each contrast administration. Results: Regression analysis revealed a good overall association (R2 = 0.81) between PP_FLM and PP_DECT, with PP_DECT substantially overestimating PP_FLM up to 30%, with limits of agreement of -18 and 18%, Low PP_FLM was underestimated, while high PP_FLM was overestimated by PP_DECT, indicating a higher sensitivity of the later. Changes of PP_DECT and PP_FLM had a concordance of 69.4% for all measurements. Agreement and concordance were highest in ventilated and lowest in non-ventilated ROIs. No persistent iodine enhancement was detected in the lung parenchyma after repetitive measurements per hour. Conclusions: Dual-energy CT based measurement of pulmonary perfusion shows promising results indicating its feasibility in translational research on strategies of mechanical ventilation.