The effects of nitric oxide (NO) donors on the L-type Ca²⁺ current (I_Ca,L) and the muscarinic activated K⁺ current (I_K,ACh) were studied in isolated rat cardiac myocytes. The nitrosothiol S-nitroso-N-acetyl-d,l-penicillamine (SNAP, 1 pm-1 µm) strongly potentiated the stimulation of the I_Ca,L elicited by subthreshold concentrations of isoprenaline (Iso, 0.1-0.5 nm) in ventricular myocytes. The effect of SNAP was mimicked by 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO, 1 pm-1 nm), a NONOate, and was blunted by carboxy-2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (100 µm), a NO trap. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (10 µm), a guanylyl cyclase inhibitor, did not alter the effect of SNAP. SNAP (1 pm-1 µm) did not modify the effect of L858051 (0.1-0.3 µm), a forskolin analogue that activates adenylyl cyclase, on I_Ca,L and did not enhance the basal I_Ca,L in the presence of rolipram (1 µm), a phosphodiesterase type 4 inhibitor. Superfusion with Rp-CPT-cAMPS (500 µm), or internal dialysis with cAMP-dependent protein kinase (cA-PK) inhibitory peptide (PKI; 20 µm), inhibitors of the ca-PK, blunted the effect of SNAP (1 nm and 1 µm) on the Iso-stimulated (1-100 pm) I_Ca,L. SNAP (1 nm and 1 µm) potentiated the threshold stimulation of I_Ca,L elicited by internal GTP-S (10 µm), a non-hydrolysable analogue of GTP. SNAP (1 pm-1 µm) and DEANO (1 µm) potentiated the stimulation of I_K,ACh elicited by low concentrations of ACh (1-2 nm) in rat atrial myocytes. The threshold stimulation of I_K,ACh elicited by internal guanylyl imidodiphosphate (10 µm) was also potentiated by NO donors. SNAP (1 µm) did not modify I_K,ACh reconstituted in human embryonic kidney 293 cells, in the absence or in the presence of ACh (1 or 10 nm). Taken together, these data suggest that NO is a cGMP-independent modulator of G-protein-coupled muscarinic and ß-adrenergic receptor actions on cardiac ion channels. Although this action of NO seemed to occur at the level of G proteins, it appeared to require a component distinct from receptors, G proteins or their effectors.