Species differences in response to hypoxic damage have been observed in studies using whole hearts. The aims of this study were to determine whether (i) species differences in response to simulated hypoxia could be detected at the level of the single myocyte, and (ii) there were any interspecies differences in the Ca2+ handling properties of the cells. Ventricular myocytes were isolated from hearts of adult rats and guinea-pigs and electrically stimulated on the stage of a fluorescence microscope. Cell length was measured using an edge-tracking device, and total intracellular [Ca2+] ([Ca2+]i) determined using indo-1. Cells were exposed to metabolic inhibition (MI) (2.5 mM NaCN and no glucose) to simulate hypoxia followed by washout of CN and re-addition of glucose ('reperfusion'). Following exposure to MI, rat cells underwent rigor contracture in 18.8 ± 0.8 min (n = 80 cells), whereas the time was longer for guinea-pig cells (32.9 ± 1.2 min, n = 83) (P < 0.001). If cells were reperfused after 1-5 min in rigor, then rat cells showed improved morphological recovery compared with guinea-pig cells (P < 0.05); thereafter recovery decreased with increasing time spent in rigor, and was similar in both groups. In indo-1 loaded cells, [Ca2+]i was significantly increased in cells from both species at the end of MI; however, the actual increase was much higher in guinea-pig cells. Upon reperfusion, [Ca2+]i recovered fully in rat cells, but in guinea-pig cells there was no significant decrease. The restoration of [Ca2+]i to normal levels in rat cells following MI was associated with improved contractile recovery compared with guinea-pig cells. We conclude that rat cells are more resistant to effects of MI than are guinea-pig cells; this may be related to species differences in Ca2+ handling during and following exposure to MI. Experimental Physiology (2000) 85.5, 505-510.