The positive contractile effect of nitric oxide (NO) donors was studied on isolated rat ventricular cardiomyocytes within a range of a positive force/frequency relationship. We determined whether the observed effect depended on cGMP. The NO donors S-nitroso-acetyl-D,L-penicillamine (SNAP) and N-[4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl]-1,3-propanediamine (spermine-NONO) increased contractile responsiveness transiently in a concentration- and frequency-dependent manner. The influence of NO donors on cGMP levels was enhanced under beating conditions. The positive contractile effect of NO donors was inhibited by adenosine 3',5'-cyclic monophosphothioate Rp diastereomer (Rp-cAMPS), but not by bisindolylmaleimide. Inhibition of the soluble guanylyl cyclase (sGC) by 1 H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ) inhibited the positive contractile effect of NO donors. Direct activation of sGC by 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC1) or addition of 8-bromo cGMP increased cell contractility comparably to NO donors. Inhibition of G(alphas) proteins by NF441 inhibited the positive contractile effect of NO donors. In contrast, NO donors did not potentiate the positive contractile effect of forskolin. These results demonstrate that the positive contractile effect of NO donors on rat ventricular cardiomyocytes working in a range of a positive force/frequency relationships is enhanced. It is mediated by NO-dependent stimulation of the sGC interacting with G(alphas) proteins.