Changes in action potential duration (APD) occur following a change in heart rate. The time course of these changes has two distinct initial phases; i) a rapid component occurring immediately upon a change of rate and, ii) a slower component occurring over a period of tens to hundreds of seconds. We have previously reported a hysteresis between the change of intracellular calcium ([Ca2+]i) and APD1, which suggests that the slow change of APD is not dependent on [Ca2+]i. Our aim here therefore, was to determine if the slow change in APD would correlate with changes in intracellular sodium ([Na+]i). Sheep were killed in accordance with The Home Office Animal (Scientific Procedures) Act 1986 for enzymatic isolation of left ventricular mid myocardial myocytes. Myocytes loaded with the sodium indicator SBFI were current clamped via perforated patch and paced at 0.25 Hz, 1 Hz and 0.25 Hz sequentially, to steady state APD and SBFI fluorescence values. On increasing pacing frequency from 0.25 to 1 Hz the rate constant of APD90 change was 0.062 ± 0.024 s-1 whilst the rate constant of change of APD90 on returning to 0.25 Hz was 0.032 ± 0.011 s-1. These values agree with those we reported previously1. However, these changes in pacing frequency did not result in any consistent change in SBFI ratio. Furthermore, a more abrupt frequency change from 0.25 to 4 Hz also failed to produce a detectable change of SBFI ratio. To demonstrate that we were able to detect changes in [Na+]i we adopted two approaches; i) in un-stimulated cells application of ouabain (1-5 mM) and nickel (5 mM) was used to block both the sodium-potassium pump and sodium-calcium exchanger and, ii) we stimulated cells in the presence of ouabain. Both approaches evoked a detectable increase of SBFI ratio. From these data, and those observed previously1, we suggest that the change in APD upon change of pacing frequency is neither dependent on a change in [Ca2+]i or a change of bulk [Na+]i.