We have investigated myosin isoform expression during progressive cardiac hypertrophy and the development of congestive heart failure in young male rats. Cardiac enlargement was produced by placing a constricting band (0.024-inch diameter) around the ascending aorta of 25-day-old animals, which resulted in progressively increased stenosis as the rat matured. A 57% and 77% cardiac hypertrophy was observed at 2 and 8 weeks, respectively, with signs of congestive failure at the latter time point. Myosin isoform expression was examined in the subendocardial and subepicardial myocardium of the left ventricle and the free wall of the right ventricle by use of native gel electrophoresis. The percentage of the V3 isoform increased dramatically in both ventricles. In the subendocardial myocardium of the left ventricle, expression of the V3 isoform increased (p<0.05) relative to the subepicardial myocardium at 2, 4, and 8 weeks (17.1% vs. 10.2%, 29.4% vs. 18%, and 46.6% vs. 36.2%). In addition to regional differences within a given transmural segment, we also observed a high degree of heterogeneity in myosin isoform expression throughout a given layer (particularly the subendocardial myocardium) when small (≤10-15 mg) adjacent samples were examined. This variability illustrated a potential danger in interpretation of gel results obtained from a single small tissue sample. Thus, cardiac hypertrophy produced by pressure overload in 25-day-old rats resulted in significantly increased V3 myosin in both the left and right ventricles. Furthermore, within the hypertrophied left ventricle, the subendocardial myocardium contained a significantly greater percentage of V3 myosin than the subepicardial myocardium. These results demonstrate that in terms of myosin isoform expression in the rat heart, the subepicardial and subendocardial myocardium do not respond to pressure overload in an identical manner.