Nitric oxide (NO) is constitutively generated by cardiac myocytes and has important roles in cardiac function, including modifying L-type Ca2+ currents (ICa,L). The precise nature of this modification remains elusive with NO reported to increase, reduce or have biphasic effects on ICa,L (Tamargo et al., 2010). Here we explore the effects of NO signalling on ICa,L recorded from enzymatically dissociated guinea pig ventricular myocytes. To optimize cytoplasmic signalling we used a perforated whole-cell switched voltage-clamp technique, achieved by including 25μM β-escin in the pipette solution. All experiments were done at 36 ± 1°C and in the presence of the nitric oxide synthase inhibitor L-NAME (20 µM) to inhibit endogenous NO production. Isoprenaline (Iso, 100 nM) increased peak ICa,L at 0 mV by 2.10 ± 0.09 (mean ± s.e.m., n = 10) normalized to control. Subsequent addition of the NO donor SNAP (S-nitroso-N-acetyl-DL-penicillamine, 100 μM) decreased this to 1.47 ± 0.13 (n = 10, P < 0.05 one way ANOVA). These concentrations of Iso and SNAP were used throughout. Application of SNAP alone had little effect on basal ICa,L. Activation curves, obtained by simultaneous fitting of GHK and Boltzmann functions to current-voltage curves, gave values for V1/2, the membrane potential of half-maximal activation, of -2.8 ± 1.8, -12.4 ± 1.8 and -12.7 ± 2.0 mV (n = 10) in control, Iso and Iso + SNAP respectively; their corresponding slopes (k) were 12.3 ± 0.7, 12.1 ± 0.1 and 12.1 ± 0.6 mV. Although SNAP reduced the Iso enhanced ICa,L it did not reverse the negative shift of the activation curve. Application of NAP (N-acetyl-D-penicillamine), an analogue of SNAP which does not release NO, had no effect on Iso enhanced ICaL. To test whether the SNAP released NO acts on ICa,L by activating soluble guanylyl cyclase (sGC) we repeated these experiments in the presence of ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), a sGC inhibitor. SNAP inhibition of Iso enhanced ICa,L was maintained in the presence of ODQ (25 µM). Peak currents normalized to control values at 0 mV were 1.78 ± 0.15 and 0.92 ± 0.14 in Iso + ODQ and Iso + ODQ + SNAP respectively (P < 0.05, n = 5). Direct activation of sGC independently of NO by BAY 60-2770 (1 µM), however, gave results similar to those observed with SNAP. Peak currents normalized to control values at 0 mV were 1.82 ± 0.18 and 1.26 ± 0.13 in Iso and Iso + BAY 60-2770 respectively (P < 0.05, n = 4). Thus direct activation of sGC mimics the effect of SNAP induced NO release, yet inhibiting sGC does not remove the SNAP induced reduction of Iso enhanced ICa,L. These results suggest that NO modulates ICa,L through more than one mechanism.