—Nitric oxide (NO) donors were recently shown to produce biphasic contractile effects in cardiac tissue, with augmentation at low NO levels and depression at high NO levels. We examined the subcellular mechanisms involved in the opposing effects of NO on cardiac contraction and investigated whether NO modulates contraction exclusively via guanylyl cyclase (GC) activation or whether some contribution occurs via cGMP/PKG-independent mechanisms, in indo 1–loaded adult cardiac myocytes. Whereas a high concentration of the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 100 μmol/L) significantly attenuated contraction amplitude by 24.4±4.5% (without changing the Ca²⁺ transient or total cAMP), a low concentration of SNAP (1 μmol/L) significantly increased contraction amplitude (38±10%), Ca²⁺ transient (26±10%), and cAMP levels (from 6.2 to 8.5 pmol/mg of protein). The negative contractile response of 100 μmol/L SNAP was completely abolished in the presence of the specific blocker of PKG KT 5823 (1 μmol/L); the positive contractile response of 1 μmol/L SNAP persisted, despite the presence of the selective inhibitor of GC 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μmol/L) alone, but was completely abolished in the presence of ODQ plus the specific inhibitory cAMP analog Rp-8-CPT-cAMPS (100 μmol/L), as well as by the NO scavenger oxyhemoglobin. Parallel experiments in cell suspensions showed significant increases in adenylyl cyclase (AC) activity at low concentrations (0.1 to 1 μmol/L) of SNAP (AC, 18% to 20% above basal activity). We conclude that NO can regulate both AC and GC in cardiac myocytes. High levels of NO induce large increases in cGMP and a negative inotropic effect mediated by a PKG-dependent reduction in myofilament responsiveness to Ca²⁺. Low levels of NO increase cAMP, at least in part, by a novel cGMP-independent activation of AC and induce a positive contractile response.