A new color Doppler myocardial imaging (CDMI) system with high spatial and temporal resolution and novel postprocessing modalities has been developed that could allow quantifiable stress echocardiography. The purpose of this study was to determine whether regional myocardial systolic velocities could be accurately and reproducibly measured both at rest and during bicycle ergometry by using CDMI. Thirty normal subjects were examined with CDMI at rest, and peak mean systolic myocardial velocities (MSV) were measured for 34 predetermined left ventricular myocardial segments. Interobserver variability and intraobserver variability were established for all segments. Submaximal bicycle ergometry was performed in 20 normal subjects by using standardized weight-related increases in workload. MSV were measured at each step of exercise for 16 left ventricular stress echo segments. At rest, a base-apex gradient in regional MSV was recorded with highest longitudinal shortening velocities at the base. A similar pattern was noted for circumferential shortening MSV. Measurements were predictable and highly reproducible with low interobserver and intraobserver variability for 26 of 34 segments. Reproducibility was poor for basal anteroseptal segments in all views and mid anterior, anteroseptal, and septal segments in the short-axis views. During exercise, mid and basal segments of all walls showed a significant increase of MSV between each workload step and for apical segments between alternate steps. The resting base-apex velocity gradient observed at rest remained in all walls throughout ergometry. Thus a CDMI system with improved spatial and temporal resolution and postprocessing analysis modalities provided reproducible and accurate quantification of segmental left ventricular circumferential and longitudinal contraction both at rest and during exercise.