Aims We investigated the role of ventricular repolarization sequence in ventricular diastolic function. Methods and results Arterially perfused canine left ventricular wedge preparation with simultaneous recording of action potentials and isometric contractile force was used to establish the relationship between ventricular repolarization and relaxation sequences. An isolated rabbit working heart model was used to investigate role of ventricular repolarization sequence in ventricular diastolic function. Under controlled conditions, similar to transmural dispersion of repolarization (TDR), there existed a time difference between initiation of epicardial and endocardial relaxation (TREpi–Endo, 47.4 ± 13.9 ms) with epicardium relaxing earlier. There was a strong correlation between TDR and TREpi–Endo (r2 = 0.99, n = 5) and the interventions that changed transmural repolarization sequence led to parallel changes in transmural relaxation sequence. In isolated rabbit working hearts, reversal of the transmural repolarization sequence that manifested as negative T wave was associated with a significant increase in isovolumic relaxation time (from 49.2 ± 19.1 to 76.4 ± 12.1 ms, n = 5, P = 0.001). Conclusion There is a strong correlation between transmural repolarization and relaxation sequences. A positive T wave that denotes transmural repolarization sequence from epicardium to endocardium is essential for normal diastolic function of ventricle and the reversal of such sequence is associated with ventricular diastolic dysfunction.