Objective:l-Ascorbic acid has been described to exert multiple beneficial effects in cardiovascular disorders associated with impaired nitric oxide (NO)/cGMP signalling. The aim of the present study was to investigate the effect of vitamin C on the most prominent physiological target of endogenous and exogenous NO, i.e. soluble guanylyl cyclase (sGC). Methods: To address this issue we used a highly purified enzyme preparation from bovine lung (from the slaughterhouse). Enzymic activity was measured by a standard assay based on the conversion of [α-32P]GTP to [32P]cGMP and the subsequent quantification of the radiolabelled product. NO was quantified using a commercially available Clark-type electrode. Results: Stimulation of sGC by the NO donor 2,2-diethyl-1-nitroso-oxyhydrazine was inhibited by ascorbate with an IC50 of ∼2 μM. Maximal enzyme inhibition (∼70%) was observed at 0.1–1 mM vitamin C. Stimulation of sGC by the NO-independent activator protoporphyrin-IX was also inhibited with similar potency. The effect of ascorbate on sGC was largely antagonised by reduced glutathione (1 mM) and the specific iron chelator diethylenetriaminepentaacetic acid (0.1 mM). Electrochemical experiments revealed that NO is potently scavenged by vitamin C. Consumption of NO by ascorbate was prevented by reduced glutathione (1 mM), diethylenetriaminepentaacetic acid (0.1 mM) and superoxide dismutase (500 units/ml) whereas up to 5000 units/ml superoxide dismutase failed to restore sGC activity. Conclusions: Our results suggest that physiological concentrations of l-ascorbic acid diminish cGMP accumulation via both scavenging of NO and direct inhibition of sGC.