The molecular mechanisms associated with the effects of various pathological stimuli on myocardial tissue, as well as the mechanisms by which Ca2+ antagonists exert their protective effect, are poorly understood. With the use of digital image processing of Fura-2 fluorescence, we have shown that the mean intracellular free Ca2+ concentration of single isolated rat cardiomyocytes is increased upon exposure to various pathological stimuli (high extracellular Ca2+, veratrine). This increased Ca2+ content coincided with an increased number of hypercontracted cells. Pretreatment with flunarizine under these experimental conditions lowered the free intracellular Ca2+ concentration, thereby reducing the number of hypercontracted cells. Verapamil had no effect. The kinetics of changes in intracellular Ca2+ in electrically paced cardiomyocytes were not affected by flunarizine, but were significantly altered by the beta agonist isoprenaline. In addition, isoprenaline increased the mean diastolic intracellular free Ca2+ concentration of paced cardiomyocytes, whereas it remained unchanged in flunarizine treated cells. We conclude that flunarizine reduces intracellular free Ca2+ levels in isolated cardiomyocytes under pathological conditions, but does not affect physiological processes mediated by Ca2+. The report also illustrates the possibilities of digital imaging microscopy in the study of ion distributions in living cells.