The present study investigated the effects of gender on intracellular [Ca2+] ([Ca2+]i) in freshly isolated rat cardiac myocytes. Changes in [Ca2+]i in response to varied extracellular [Ca2+], different stimulus frequencies and addition of caffeine and isoprenaline were monitored using fura-2 in both male and female cardiac myocytes. Increasing extracellular [Ca2+] and stimulus frequency resulted in significant increases in peak [Ca2+] and the amplitude of the Ca2+ transient in both male and female cardiac myocytes. However, as extracellular [Ca2+] was raised, peak [Ca2+] and the amplitude of the Ca2+ transient increased significantly more in male than female cardiac myocytes. In addition a significant difference between male and female cells at each stimulus frequency was apparent. The time course of decay of the Ca2+ transient was significantly slower in female cardiac myocytes when compared with male cardiac myocytes, along with significantly slowed times to peak shortening and 50% relaxation, and a reduced extent of shortening. There was no significant difference in the amplitude of caffeine-induced [Ca2+]i responses between male and female cells, however, [Ca2+]i increased more readily in male cells than in female cells when isoprenaline was added. The data demonstrate that, under a variety of conditions, intracellular [Ca2+] rises to higher levels in cardiac myocytes from male as compared to female rats.