Terfenadine and ketoconazole are the most widely used positive reference agents in non-clinical cardiac repolarization safety studies. The aim of the present study was to evaluate the effects of terfenadine, ketoconazole and their combination on QT prolongation using conscious guinea pigs. Conscious telemetered guinea pigs were orally administered terfenadine (50 mg/kg), ketoconazole (200 mg/kg) or a combination of the two, and effects on QT were recorded using a telemetry system. The QT correction was carried out with Bazett's formula to eliminate confounding effect of HR. Neither terfenadine nor ketoconazole produced any effect on the RR and QT intervals, QRS complex or heart rate (HR). However, a combination of terfenadine and ketoconazole significantly prolonged the RR and QT intervals and decreased HR in a time-dependent manner. This study demonstrated that the combination of terfenadine and ketoconazole produces QT prolongation in conscious telemetered guinea pigs.