The cardiac action potential results from a complex interplay of voltage-dependent ion channels, pumps and transporters. In this study we have used a fast potentiometric dye to record changes in the morphology of the action potential in response to cisapride, which is known to cause action potential prolongation. Ventricular myocytes, isolated from humanely killed adult guinea pigs, were loaded with 5 uM di-8-ANEPPS for 20 min. Switch clamp experiments, using a patch electrode, were used to confirm that changes in the fluorescence emission ratio from di-8-ANEPPS had a linear relationship with membrane potential between −80 mV and + 60 mV. Averages of 20 traces of optical recordings were used to increase signal: noise ratio. Control values of the action potential duration to 90% repolarization (APD₉₀) measured optically (355 ± 11 ms, mean ± S.E.M) were significantly longer in duration than those recorded using a microelectrode (301 ± 14 ms; p<0.01, n = 10, Student’s t test). Acute perfusion of 5 uM di-8-ANEPPS shows a maximal increase in APD₉₀ of 16 ± 4%, suggesting that the increase in APD observed during optical recording may result from the loading of the dye. Comparisons between action potentials recorded simultaneously both optically and using a patch electrode reveal that the optical measurements are essentially identical to those recorded electrically. Cisapride, which is known to block I_Kr, caused increases in APD₉₀ at concentrations of 10-300 nM. However, at 1000 nM cisapride APD₉₀ values were shorter than at 300nM, suggesting additional effects of the drug. Increases in APD₉₀ at 300nM cisapride compared to control values were 7.7 ± 2.3% when recorded optically and 10.9 ± 2.0% when recorded using a microelectrode (NS). EC₅₀ values calculated from dose-response curves were 91 ± 46 nM when recorded optically, compared to 81 ± 20 nM when recorded using an electrode (NS). These data suggest that guinea pig ventricular myocytes loaded with Di-8-ANEPPS have a longer basal APD, so that cisapride-induced action potential prolongation may be less pronounced when recording optically with this dye, when compared with recordings using a microelectrode. Therefore, optical studies using di-8-ANEPPS may underestimate the arrhythmogenic potential of compounds. However, monitoring cardiac APD in single cells with the use of a voltage-sensitive dye may be feasible for use as an assay.