Right ventricular cardiac tissue (10-20 mg wet weight) was obtained from anesthetized adult dogs by endomyocardial biopsy. The biopsy could be repeated in one dog every 2 weeks for up to 3 months. Fifty to 200 cardiomyocytes, dispersed with collagenase and trypsin, were collected by centrifugation of the cells with 50percent; polysucrose-sodium diatrizoate solution (Ficoll-Paque). Single cardiomyocytes were suspended in a minimum essential medium containing 20percent; fetal bovine serum and 8-bromoadenosine 3': 5'-cyclic monophosphate (0.1 mM) for up to 3 weeks. Approximately 70-80percent; of the cultured cardiomyocytes were rod shaped after 24 hours (10-20percent; after 7 days). Cytoplasmlc organelles of the cultured cells, examined with a transmission electron microscope, were within the normal range of canine heart morphology in vivo. Resting membrane potential of the cells was about -80 mV when superfused with a Krebs' solution containing 4.7 mM potassium ions. The action potential lasted for 300 msec and had a peak amplitude of about 120 mV. Voltage-clamp experiments demonstrated the presence of an inward calcium current (±0.9 nA at +9 mV), which was facilitated by isoproterenol (0.1-1 μM). The background potassium current showed typical inward rectification at potentials more negative than -80 mV. The results indicate that morphological, electrophysiological, and pharmacological properties of the cultured cardiomyocytes were intact. We propose that the culture techniques we have developed can be useful for repeated investigation on functional aspects of cardiac muscles in myocardial disease.