We have shown previously that extracellular cysteine is necessary for cellular responses to S-nitrosoalbumin. In this study we have investigated mechanisms involved in accumulation of extracellular cysteine outside vascular smooth muscle cells and characterized the role a cystine-cysteine release in transfer of NO-bioactivity. Incubation of cells with cystine led to cystine uptake, reduction and cysteine release. The process was inhibitable by extracellular glutamate suggesting a role for system xc- amino acid transporters. Smooth muscle cells express this transporter constitutively and induction of the light chain component, xCT, by either diethyl maleate or SIN-1 led to glutamate-inhibitable cystine uptake and an increased rate of cysteine release from cells. Likewise overexpression of xCT in smooth muscle cells or endothelial cells led to glutamate-inhibitable cysteine release. The resulting extracellular cysteine was found to be required for transfer of NO from extracellular S-nitrosothiols into cells via system L transporters leading to formation of cellular S-nitrosothiols. Cysteine release coupled to cystine uptake was also found to be required for cellular responses to S-nitrosoalbumin and facilitated S-nitrosoalbumin-mediated inhibition of EGF signaling. These data show that xCT expression can constitute a cystine-cysteine shuttle whereby cystine uptake drives cysteine release. Furthermore we show that extracellular cysteine provided by this shuttle mechanism is necessary for transfer of NO-equivalents and cellular responses to S-nitrosoablumin.