Nitric oxide (NO) signaling through the formation of cGMP is well established; however, there seems to be an increasing role for cGMP-independent NO signaling. Although key molecular details remain unanswered, S-nitrosation represents an example of cGMP-independent NO signaling. This modification has garnered recent attention as it has been shown to modulate the function of several important biochemical pathways. Although an analogy to O-phosphorylation can be drawn, little is known about protein nitrosothiol regulation in vivo. In solution, NO readily reacts with oxygen to yield a nitrosating agent, but this process alone provides no specificity for nitrosation. This lack of specificity is exemplified by the in vitro poly-S-nitrosation of caspase-3 (Casp-3, ref. 6) and the ryanodine receptor. Previous in vivo work with Casp-3 suggests that a protein-assisted process may be responsible for selective S-nitrosation of the catalytic cysteine (Cys163). We demonstrated that a single cysteine in thioredoxin (Trx) is capable of a targeted, reversible transnitrosation reaction with Cys163 of Casp-3. A greater understanding of how S-nitrosation is mediated has broad implications for cGMP-independent signaling. The example described here also suggests a new role for Trx in the regulation of apoptosis.