The Na+-Ca2+ exchanger (NCX) is up-regulated in the neonatal rabbit heart. Because the duration of membrane depolarization is an important determinant of calcium entry via NCX, pharmacological agents that lengthen the action potential (AP) may significantly increase the amount of activator calcium in newborns. We tested this potentially novel therapeutic strategy by using action potential voltage clamp steps or using dofetilide, a blocker of IKr, to prolong the action potential duration (APD). The effects of changing APD on calcium transients were determined in ventricular myocytes at different developmental stages: newborn (1-4 days), juvenile (9-10 days), and adult ventricular myocytes (35°C; 1 Hz). Calcium transient amplitude in neonatal myocytes increased substantially with clamping with longer APs. In contrast, exposure to dofetilide (0.1, 1, and 10 μM) under current clamp conditions increased APD in a concentration-dependent manner but had no significant effect on calcium transient amplitude in either neonates or adults. When the AP was held constant under voltage clamp conditions, dofetilide decreased the calcium transient amplitude in neonates. This effect is likely related to inhibition of sodium-calcium exchanger and L-type Ca2+ currents (ICa), as observed in separate experiments. These results suggest that dofetilide has a paradoxical effect on APD and calcium transients in the newborn heart.