The potency of neuropeptide Y (NPY) to cause negative and positive contractile responses in rat ventricular cardiomyocytes was investigated. In these cells, NPY was found to activate the transient outward K+ current (Ito) and the slow inward Ca2+ current (Isi). As reported before (H. M. Piper, B. C. Millar, and J. R. McDermott, Naunyn Schmiedeberg's Arch. Pharmacol. 340: 333-337, 1989), NPY attenuated the increase in the contractile response induced by isoprenaline (10(-7) M). This effect of NPY could be abolished by 1) the presence of the inhibitor of Ito, 4-aminopyridine (4-AP, 0.5 mM); 2) pretreatment of the cells with pertussis toxin (1 microgram/ml for 6 h); and 3) the presence of the 19-amino acid COOH-terminal fragment of NPY, NPY-(18-36) (10(-6) M). In the absence of isoprenaline, but in the presence of 4-AP, NPY exerted a stimulatory effect on the cardiomyocytes. This effect could be abolished 1) by using the inhibitor of the Isi, verapamil (10(-8) M), but not 2) by pretreatment with pertussis toxin, nor 3) by coincubation with NPY-(18-36). The results indicate that in the rat the antiadrenergic negative contractile effect of NPY results from its action on the Ito. Blockade of this current by 4-AP unmasks a positive contractile effect of NPY that is related to activation of the Isi.