OBJECTIVE: To measure the radial and longitudinal velocities of several myocardial segments of the left ventricular wall by use of tissue Doppler imaging (TDI) in healthy cats and determine the repeatability and reproducibility of the technique. ANIMALS: 6 healthy cats. PROCEDURE: 72 TDI examinations were performed on 4 days by the same trained observer. Radial parameters included left endocardial and epicardial myocardial velocities. Longitudinal parameters included left basal, middle, and apical myocardial velocities. RESULTS: All velocity profiles had 1 positive systolic wave (S) and 2 negative diastolic waves (E and A). Myocardial velocities were higher in the endocardial than epicardial segments during the entire cardiac cycle (systolic wave S, 4.4 +/- 0.82 and 1.9 +/- 0.55; diastolic wave E, 9.7 +/- 1.70 and 2.2 +/- 0.74; and diastolic wave A, 5.1 +/- 1.56 and 1.4 +/- 0.76, respectively). Velocities were also higher in the basal than in the apical segments (systolic wave S, 4.7 +/- 0.76 and 0.2 +/- 0.11; diastolic wave E, 9.7 +/- 1.36 and 0.5 +/- 0.17; and diastolic wave A, 3.7 +/- 1.51 and 0.2 +/- 0.13, respectively). The lowest within-day and between-day coefficients of variation were observed in endocardial segments (8.2% and 6.5% for systolic wave S and diastolic wave E, respectively) and in the basal segment in protodiastole (5.5%). CONCLUSIONS AND CLINICAL RELEVANCE: Repeatability and reproducibility of TDI were adequate for measurement of longitudinal and radial left ventricular motion in healthy awake cats. Validation of TDI is a prerequisite before this new technique can be recommended for clinical use.