We have previously reported a lengthening of the action potential of single rat sub-epicardial left ventricular myocytes (EPI) isolated from the hearts of female animals that undertook 6-7 weeks of voluntary wheel running (Natali et al. 2002). We have tested the hypothesis that a decrease in the transient outward current (Ito) might contribute to this change in action potential configuration. Ito was measured under whole cell, voltage clamp conditions at 37°C in single EPI and sub-endocardial myocytes (ENDO) isolated from hearts of humanely killed female Sprague-Dawley rats undergoing 6-7 weeks of voluntary wheel running (TRN) or their sedentary equivalents (SED). Tissue samples from these four groups were also analysed using real-time reverse transcription polymerase chain reaction to measure the mRNA expression ratios of Kv4.2, Kv4.3 and Kv1.4, genes which encode for cardiac Ito. For each transcript, 3 measurements were performed on every cDNA sample and ratios relative to 6 of the cDNA samples, which were selected as reference standards, were determined from the respective delta CT (crossing point) value and average efficiency of the reaction. These ratios were then related to the average content of 28S cDNA in each sample. Ito density was dependent upon region being significantly greater in EPI than ENDO for both SED and TRN (Ito recorded between -40 and +60mV, P<0.05, two way repeated measures ANOVA, n = 13-28 cells in each group). However, whilst there was no significant difference in the density of Ito in ENDO between TRN and SED, there was a statistically significant decrease in Ito recorded between -40 and +60mV in TRN EPI compared with SED EPI (P<0.05, two-way repeated measures ANOVA, n = 13-28 cells in each group). In addition, normalised Ito density recorded in individual TRN EPI myocytes at +40mV was found to be inversely proportional to cell size (linear regression, R=-0.529, P ENDO) but in addition was increased by voluntary exercise (3.87±0.29 vs 3.03±0.22 mRNA expression ratios in TRN EPI vs SED EPI respectively, (mean ± S.E.M.) P< 0.05, 2 way ANOVA, n=10 in each group). This may be a response to the functional decrease in Ito. Expression of Kv4.3 and Kv1.4 did not correlate with changes in Ito density. We conclude that a regionally dependent decrease in Ito current may explain the effect of voluntary exercise on the action potential configuration of single rat ventricular myocytes.