Cardiac troponin I (TnI) knockout mice exhibit a phenotype of sudden death at 17-18 days after birth due to a progressive loss of TnI. The objective of this study was to gain insight into the physiological consequences of TnI depletion and the cause of death in these mice. Cardiac function was monitored serially between 12 and 17 days of age by using high-resolution ultrasonic imaging and Doppler echocardiography. Two-dimensional B-mode and anatomical M-mode imaging and Doppler echocardiography were performed using a high-frequency (~20-45 MHz) ultrasound imaging system on homozygous cardiac TnI mutant mice (cTnI^-/-) and wild type littermates. On day 12, cTnI^-/- mice were indistinguishable from wild type in terms of heart rate, atrial and left ventricular chamber dimensions, left ventricular posterior wall thickness and body weight. By day 16-17, wild type mice showed up to a 40% increase in chamber dimensions due to normal growth, whereas cTnI^-/- mice showed increases in atrial dimensions of up to 97% but decreases in ventricular dimensions of up to 70%. Mitral Doppler analysis revealed prolonged IVRT and pronounced inversion of the mitral E/A ratio only in cTnI^-/- mice indicative of impaired LV relaxation. cTnI-/- mouse hearts showed clear signs of failure on day 17, characterized by >50% declines in cardiac output, ejection fraction and fractional shortening. B-mode echocardiography showed a profoundly narrowed tube-like LV and enlarged atria at this time. Our data are consistent with TnI deficiency causing impaired LV relaxation, which leads to diastolic heart failure in this model.