Objective: Inward remodeling of small arteries is associated with low blood flow, hypertension and chronic vasoconstriction. Previous work showed that in all three conditions, transglutaminases (Tgases) play a causal role during the remodeling process. Tgases are multifunctional proteins which act as cross-linking enzymes, affect cell binding, and cell signalling. In the present study, we addressed the localization, activation and specific function of Tgases within the vessel wall during remodeling. Methods: In vitro, mouse mesenteric arteries were exposed to Tgases, fluorescent substrate (FITC-cadaverine) and thiol- reducing compounds for 24 hours. Passive pressure- diameter relationships were determined and confocal microscopy was used to reveal Tgase activity. Results: Exogenous TG2 induced inward remodeling only under reducing conditions (15±3% vs. 2±3% decrease in lumen diameter at 80 mmHg). The active site mutant hTG2 (Zedira) did not induce remodelling (1±1%). Dithiothreitol (DTT), a cell-permeable reducing agent, induced remodeling (20±3% decrease in lumen diameter) and cross-linking activity at the cell surface of smooth muscle cells. Both the remodeling and cross-linking activity induced by DTT was blocked by the TG2 inhibitor L682.777 (Zedira), implicating endogenous TG2. The cell-impermeable reducing agent tris(2-carboxyethyl)-phosphine (TCEP), did not induce remodeling (1±1% decrease lumen diameter) and cross-linking activity. Conclusion: Intra-cellular reduction induces cell- surface Tgase activity and vascular remodeling. In the extra-cellular space, TG2 again requires a reducing environment to induce cross-linking and inward remodeling. These results suggest that reduction of thiol groups within TG2 may be an important regulator of both secretion and cross- linking activity during vascular remodeling. Supported by the Netherlands Heart Foundation, grant NHS2005.B080.