1. Nitric oxide (NO) is a potent inhibitor of platelet activation, that inhibits the agonist-induced increase in cytosolic Ca2+ concentration through both cGMP-dependent and independent pathways. However, the NO-related (NOx) species responsible for cGMP-independent signalling in platelets is unclear. We tested the hypothesis that extracellular NO, but not NO+ or peroxynitrite, generated in the extracellular compartment is responsible for cGMP-independent inhibition of platelet activation via inhibition of Ca2+ signalling. 2. Concentration-response curves for diethylamine diazeniumdiolate (DEA/NO; a spontaneous NO generator), S-nitroso-N-valerylpenicillamine (SNVP; an S-nitrosothiol) and 3-morpholinosydnonomine (SIN-1; a peroxynitrite generator) were generated in platelet-rich plasma (PRP) and washed platelets (WP) in the presence and absence of a supramaximal concentration of the soluble guanylate cyclase inhibitor, ODQ (20 microM). All three NOx donors displayed cGMP-independent inhibition of platelet aggregation in PRP, but only DEA/NO exhibited cGMP-independent inhibition of aggregation in WP. 3. Analysis of NO generation using an isolated NO-electrode revealed that cGMP-independent effects coincided with the generation of substantial levels of extracellular NO (>40 nM) from the NOx donors. 4. Reconstitution of WP with plasma factors indicated that the copper-containing plasma protein, caeruloplasmin (CP), catalysed the release of NO from SNVP, while Cu/Zn superoxide dismutase (SOD) unmasked NO generated from SIN-1. The increased generation of extracellular NO correlated with a switch to cGMP-independent effects with both NOx donors. 5. Analysis of Fura-2 loaded WP revealed that only DEA/NO inhibited Ca2+ signalling in platelets via a cGMP-independent mechanism. However, preincubation of SNVP and SIN-1 with CP and SOD, respectively, induced cGMP-independent inhibition of intraplatelet Ca2+ trafficking by the NOx donors. 6. Taken together, our data suggest that extracellular NO (>40 nM) is required for cGMP-independent inhibition of platelet activation. Plasma constituents may play an important pharmacological role in activating cGMP-independent signalling by S-nitrosothiols or peroxynitrite generators.