The objective of the present study was to investigate the effects of increasing levels of transglutaminase (TG) on rheological and baking properties of Roane (soft red winter wheat) and Sharpshooter (hard red spring wheat) flours. The TG enzyme was added at six different levels (0, 0.1, 0.25, 0.5, 1.0, 1.5% w/w). A far-inograph and a texture analyzer with an SMS/Kieffer rig were used to examine the mixing properties and extensibility of the flour doughs, respectively. Results from far-inography showed decreasing flour-water absorption with increasing TG levels. Dough development time and stability values initially increased with increasing levels of TG, but decreased at higher TG levels. Extensibility values decreased and Rmax values increased significantly with increasing TG levels. Low levels of TG had improving effects on bread quality (crust and crumb characteristics), while higher levels (1 and 1.5%) had detrimental effects. The improving effect of TG at lower addition levels was more evident for the soft wheat cultivar, Roane. Overall results indicated that TG, even at very low levels, could be successfully incorporated in the dough formulation to improve breadmaking quality.