Abstract: AIM: To evaluate the effect of cyclosporine (CsA) on the cellularity and intracellular calcium concentration ［Ca2+］i in neonatal rat cardiomyocytes. METHODS: Neonatal rat cardiomyocytes were prepared from 1- to 3-day-old neonatal Wistar rats. After CsA (1×10-3 g/L, 1×10-2 g/L, 2×10-2 g/L) was gastrically instilled into cardiomyocytes culture media randomly for 2, 4, 6, 8 and 12 h, the cellular structure and contractility were observed under phase-contrast microscope. ［Ca2+］i was measured by a laser scanning confocal microscope with excitation at 488 nm and emission at 530 nm. RESULTS: Compared with control, no obvious difference was observed in the cellularity of cardiomyocytes after incubation at different concentrations of the CsA for 2 h. ［Ca2+］i of cardiomyocytes significantly increased after incubation with CsA 2×10-2 g/L for 2 h compared with that in the other three groups (P<0.01). When the cardiomyocytes were incubated with CsA for 4 h, contractility of the cardiomyocytes was attenuated gradually and more particles were seen. ［Ca2+］i of the cardiomyocytes treated with each concentration of CsA increased compared with control (P<0.05). ［Ca2+］i of the cardiomyocytes treated with CsA 1×10-2 g/L and 2×10-2 g/L increased more significantly compared with that of cardiomyocytes treated with CsA 1×10-3 g/L. With treatment of CsA for 6 h, ［Ca2+］i of cardiomyocytes increased markedly (P<0.05) and more small vacuoles were seen in the cardiomyocytes. With treatment of CsA for 8 and 12 h and the increasing concentration of the CsA, more cardiomyocytes died. CONCLUSION: CsA impairs cardiomyocytes and attenuates contractility of cardiomyocytes in a concentration- and time-dependent manner of CsA. Impairment of the cardiomyocytes may be related to the effect that increased ［Ca2+］i of cardiomyocytes.