Perhaps as many as 100 proteins have been linked to S-nitrosation in vivo, but factors concerning the mechanism of formation and physiological roles for such modifications remain uncertain. We have developed a model system to address this topic using HT1080 cells, a fibrosarcoma cell line. We have integrated into these cells inducible nitric oxide synthase (iNOS) under tetracycline regulation (tet-on), and soluble guanylate cyclase (sGC), which responds to NO and possibly S-nitrosation. We are also examining thioredoxin and S-nitrosoglutathione reductase in these cells, two proteins central to S-nitroso (SNO) signaling and metabolism. The cells display increased migration in response to NO production, of possible importance for cancer metastasis. Our in vivo studies are supported with in vitro biochemical and structural studies. Progress has been best with human thioredoxin (hTrx), which has five cysteines, is important in maintaining a reducing cytosol, distributing SNO groups, and regulating cell growth and cell death. Here, we describe formation of S-nitrosated human thioredoxin (hTrx) via transnitrosation with S-nitroso-glutathione (GSNO) and describe the structure of the protein at 1.65 Å resolution, which reveals a planar, buried SNO moiety. We also describe S-nitrosation of sGC both in vivo and by transnitrosation with hTrx-SNO, which, surprisingly, may stimulate sGC catalysis.