We have previously described detailed procedures for isolating rat and canine myocytes from adult ventricular tissue, and have investigated ultrastructural and functional properties of these cells. The effects of isolation on cell morphology were tested by comparative scanning (SEM) and transmission electron microscopy (TEM) of adult canine cardiac myocytes. Cells cleanly separated at the lateral sarcolemmal borders and at the intercalated discs. Since the cells were contracted during fixation, SEM of the lateral cell borders showed longitudinal folds and transverse ridges. There were rows of regular protrusions corresponding to the mitochondria immediately beneath the sarcolemmal surface. Openings in the sarcolemmal membrane occurred at regular intervals at the level of the Z-bands and may represent the sites of externalization of the transverse tubular system. By SEM, the intercalated disc junction appeared as a microvillar border and this corresponded directly to the scalloped appearance seen by TEM. A controlled sonication procedure was developed to allow specific removal of the sarcolemma without disruption of underlying organelles. This allowed visualization of the cellular organization of myofibrils, mitochondria and the transverse tubular system. These cells retain biochemical and enzymatic characteristics of adult dog cardiac tissues such as ouabain and insulin sensitivity, fatty acid and lipoprotein uptake and metabolism, and prostaglandin synthesis. This cytological preparation permits the correlation of these types of metabolic studies with the known functions of isolated subcellular organelles of the myocardium.