Neuropeptide Y (NPY) is the most abundant peptide in the mammalian heart, but its cardiac actions are not fully understood. Here we investigate the effect of NPY in intracellular Ca²⁺ release, using isolated rat cardiac myocytes and confocal microscopy. Cardiac myocytes were field-stimulated at 1 Hz. The evoked [Ca²⁺]_i transient was of higher amplitude and of faster decay in the presence of 100 nM NPY. Cell contraction was also increased by NPY. We analyzed the occurrence of Ca²⁺ sparks and their characteristics after NPY application. NPY significantly increased Ca²⁺ sparks frequency in quiescent cells. The Ca²⁺ spark amplitude was enhanced by NPY but the other characteristics of Ca²⁺ sparks were not significantly altered. Because cardiac myocytes express both Y₁ and Y₂ NPY receptors, we repeated the experiments in the presence of the receptor blockers, BIBP3226 and BIIE0246. We found that Y₁ NPY receptor blockade completely inhibited NPY effects on [Ca²⁺]_i transient. PTX-sensitive G-proteins and/or phospholypase C (PLC) have been invoked to mediate NPY effects in other cell types. We tested these two hypotheses. In PTX-treated myocytes NPY was still effective, which suggests that the observed NPY actions are not mediated by PTX-sensitive G-proteins. In contrast, the increase in [Ca²⁺]_i transient by NPY was completely inhibited by the PLC inhibitor U73122. In conclusion, we find that NPY has a positive inotropic effect in isolated rat cardiac myocytes, which involves increase in Ca²⁺ release after activation of Y₁ NPY receptor and subsequent stimulation of PLC.