Myocardial fiber orientation undergoes an orderly transition from the epicardium to the endocardium in the left ventricle, with circumferential fibers predominating in the middle one-third of the heart wall. How fibers lying at different depths in the myocardium, running in different directions, interact to produce local deformation is not known. To define the relationship between the orientation of uniaxial myocardial fibers and local wall motion, we placed three sets of ultrasonic dimension gauges in the middle one-third of the apex-to-base distance of the left ventricle of nine dogs. One pair was placed in line and two intentionally out of line with the presumed local fiber direction. The relative angle between the gauge and the local myofibers was determined by the use of postmortem radiography and histological techniques. Our results show that in the midwall of the left ventricle, myocardial segment shortening is maximal in the direction of local fibers; the shortening measured by gauges placed out of line with the local fiber axis by more than 30 degrees was significantly less than the actual in-line fiber shortening which occurred. This suggests that functional tethering between midwall fibers and endocardial or epicardial fibers does not play a major role in the pattern of midwall deformation. We also documented that an external reference line can be used to predict midwall myofiber direction. Using this line as a guide, ultrasonic dimension gauges could be placed within an average of 12 degrees (range: 0.5 to 18.5 degrees) from the local fiber axis.