A technique has been developed for isolating a high yield of Ca2+-tolerant rod-shaped myocytes from the right and left ventricles of cat myocardial tissue. Myocytes were prepared by retrograde perfusion of the coronary arteries via the aorta with a nominally Ca2+-free (20-30 microM) modified Krebs-Henseleit buffer containing 0.12% collagenase. After exposure to physiological levels of Ca2+ (1-2.5 mM), the cells retained rod-shaped morphology, exhibited clear cross striations, and excluded the dye trypan blue (0.4%). Initial percents of viable Ca2+-tolerant rod-shaped cells were 58.6 +/- 3.4 (SE) and 51.8 +/- 3.5 for right and left ventricular cells, respectively. Viability studies demonstrated that these values decreased approximately 10% at the conclusion of a 2-h incubation in 1 mM Ca2+. The total numbers of rod-shaped myocytes obtained were 4.48 X 10(7) and 3.89 X 10(7) in nominal (8-10 microM) and 1 mM Ca2+-containing buffer, respectively. A total of 3.44 +/- 0.40 X 10(6) rod-shaped Ca2+-tolerant myocytes was initially isolated per gram of tissue wet weight. Measurements of cell length, width, and sarcomere length demonstrated no significant differences between right and left ventricular cells suspended in nominal (8-10 microM) and 1 mM Ca2+-containing buffer. No significant difference was found in the percent of binucleate cells when right and left ventricular myocytes were compared. These results demonstrate that a stable population of Ca2+-tolerant myocytes with similar morphological characteristics can be isolated from the right and left ventricles of cat myocardium.