The relative contributions of the sarcoplasmic reticulum (SR), the L-type Ca²⁺ channel and the Na⁺/Ca²⁺ exchanger (NCX) were assessed in turtle ventricular myocytes using epi-fluorescent microscopy and electrophysiology. Confocal microscopy images of turtle myocytes revealed spindle shaped cells which lack T-tubules and have a large surface area-to-volume ratio. Myocytes loaded with the fluorescent Ca²⁺ sensitive dye Fura-2 elicited Ca²⁺ transients which were insensitive to ryanodine and thapsigargin, indicating the SR plays a small role in the regulation of contraction and relaxation in the turtle ventricle. Sarcolemmal Ca²⁺ currents were measured using the perforated-patch voltage-clamp technique. Depolarising voltage steps to 0 mV elicited an inward current that could be blocked by nifedipine, indicating the presence of Ca²⁺ currents originating from L-type Ca²⁺ channels (I_Ca). The density of I_Ca was 3.2 ± 0.5 pA pF^-1, which lead to an overall total Ca²⁺ influx of 64.1 ± 9.3 µM L^-1. NCX activity was measured as the Ni⁺-sensitive current at two concentrations of intracellular Na⁺ (7 and 14 mM). Total Ca²⁺ influx through the NCX during depolarising voltage steps to 0 mV was 58.5 ± 7.7 µmol L^-1 and 26.7 ± 3.2 µmol L^-1 at 14 and 7 mM intracellular Na⁺ respectively. In the absence of the SR and L-type Ca²⁺ channels, the NCX is able to support myocyte contraction independently. Our results indicate turtle ventricular myocytes are primed for sarcolemmal Ca²⁺ transport, and most of the Ca²⁺ used for contraction originates from the L-type Ca²⁺ channel.