Three independent methods were evaluated in an effort to obtain reliable values for myocyte size in adult Sprague-Dawley rats. Cell volume was determined from isolated myocytes by a Coulter Channelyzer system. Cell volume was also determined from the product of length and cross-sectional area of isolated myocytes. Additionally, myocyte cross-sectional area was measured morphometrically from electron micrographs of whole perfusion-fixed tissue. A major goal was to determine if anatomical methods used to measure cell volume produce values comparable to the more expeditious and objective Coulter Channelyzer method. The results of these experiments showed that myocyte dimensions obtained from all three techniques were similar. The second major objective was to use the above-mentioned techniques to evaluate regional differences in myocyte size. Myocyte cross-sectional area and volume were significantly larger in the endomyocardium than in the epimyocardium of the left ventricle. Right ventricle myocytes had significantly smaller volumes and cross-sectional areas than did left ventricle myocytes. There were no regional differences in cell lengths. We conclude that the Coulter Channelyzer system gives values for isolated myocyte volume that are similar to values obtained with histometric techniques; values for isolated myocyte cross-sectional area were representative of values obtained from myocytes in whole-sectioned tissue; significant regional differences in myocyte size are present in adult rat hearts; and regional variations in myocyte size are due to differences in myocyte cross-sectional area rather than cell length.