OBJECTIVE To establish prospectively a database of normal three-dimensional systolic and diastolic endocardial and epicardial velocity values for all myocardial segments in healthy volunteers by using cine phase-contrast velocity magnetic resonance imaging, also called tissue phase mapping (TPM). METHODS The study was approved by the institutional ethics committee and was conducted according to principles of the Declaration of Helsinki; each subject provided informed written consent. Ninety-six healthy volunteers (57 [59%] men, 39 [41%] women; mean age, 38 years +/- 12 [standard deviation]) underwent cardiac phase-contrast imaging with a black blood segmented k-space gradient-echo sequence for the analysis of three-dimensional myocardial velocity with high spatial resolution at 1.5 T on basal, midventricular, and apical short-axis views. Eighteen consecutive volunteers were imaged twice to determine interstudy reproducibility, and intra- and interobserver variability values were analyzed. Systolic and diastolic velocity curves were analyzed for peak velocity and time to peak velocity in the radial, circumferential, and longitudinal directions, as well as for torsion rate and longitudinal strain rate. Mixed-effects models with a random intercept for volunteers were used to test differences among the three ventricular sections and the transmural, endocardial, and epicardial parameters. RESULTS TPM enabled reproducible assessment of myocardial velocity with small intra- and interobserver variability values. Systolic peak radial velocity was lowest at the apical level (P < .001); diastolic peak radial velocity was similar at all three myocardial levels (P = .73). As viewed from the apex, a relative counterclockwise rotation during systole was followed by a relative clockwise rotation of the apex against the base. Diastolic and systolic peak longitudinal velocity values decreased from base to apex (P < .001). A gradient between endocardium and epicardium was observed for radial velocity values, with greater endocardial velocity values (P < .001). CONCLUSIONS TPM is a reproducible comprehensive modality for assessment of regional wall motion, and intra- and interobserver variability values are low.