Technical Abstract: Grain texture affects many milling characteristics and end-use qualities in wheat (Triticum aestivum L.) such as milling yield, flour particle size, and starch damage. In wheat, grain texture is controlled primarily by the two genes puroindoline a (pina) and puroindoline b (pinb) that reside at the Hardness (Ha) locus. Variation in puroindoline activity and abundance is known to influence a variety of milling and baking traits, and the reconstitution of flour with puroindoline A protein (PINA) has been shown to positively affect loaf volume and crumb grain. To investigate which milling and baking traits are affected by the addition of puroindolines in vivo, we transformed the hard red spring wheat cultivar Hi-Line, with genetic constructs driving the expression of pina-D1a, pinb-D1a, or both pina-D1a and pinb-D1a. Transgenic lines exhibited decreased grain hardness and increased puroindoline content. Selected T3 lines were grown in replicated field trials under dry and irrigated conditions. Harvested grain was then milled and baked. Lines transformed with the puroindolines exhibited decreased total flour yields and increased break flour yields, yielding flour with lower protein and ash content. Decreases in loaf volume, mixograph absorption, and crumb grain scores were also observed in transgenic lines having high puroindoline expression. Decreased loaf volume was also observed in whole wheat bakes of transgenic soft wheats vs. normal hard wheat, indicating a direct effect of puroindolines on loaf volume. Puroindoline content did not affect dough mixing times. These results demonstrate that the puroindolines can be used to profoundly influence a variety of milling and bread baking traits in wheat.