Aims Assessing reversibility of pulmonary vascular changes through vasoreactivity testing (VRT) optimizes end-stage heart failure patient selection for heart transplant. All efforts should be made to unload the left ventricle and reduce pulmonary vascular resistance to effectively exclude irreversible pulmonary hypertension. Methods and results We reviewed our centre's cardiac transplant registry database (2009-2017) for VRT and compared haemodynamic responses with 40 ppm inhaled NO (n = 14), 14-17 μg inhaled iloprost (n = 7), and 24 h 0.1 μg/kg/min intravenous levosimendan (n = 14). Response to levosimendan was assessed by repeat right heart catheterization within 72 h. Baseline clinical and haemodynamic features were similar between groups. VRT was well tolerated in all patients. All drugs effectively reduced pulmonary artery pressures and transpulmonary gradient while increasing cardiac index, although levosimendan had a greater impact on cardiac index increase (P = 0.036). Levosimendan was the only drug that reduced pulmonary artery wedge pressure (P = 0.004) and central venous pressures (P < 0.001) and increased both left and right ventricular stroke work indexes (P = 0.020 and P = 0.042, respectively) and cardiac power index (P < 0.001) compared with NO and iloprost. Right ventricular end-diastolic pressures and central venous pressure were only decreased by levosimendan. The rate of positive responses (≥10 mmHg decrease or final mean pulmonary artery pressure ≤40 mmHg with increased/unaltered cardiac index) was lower with inhaled iloprost (14%) than with either levosimendan or NO (71% and 64%, respectively; P < 0.05). Conclusions Levosimendan may be a safe and effective alternative for pulmonary hypertension reversibility assessment or a valuable pre-test medical optimization tool in end-stage heart failure patient assessment for heart transplantation offering extended haemodynamic benefits. Whether it increases the rate of positive responses or allows a better selection of candidates to heart transplantation remains to be established.