Celiac sprue is a disease in humans that is characterized by small intestinal mucosal injury and malabsorption. Dietary exposure to gliadin and similar proteins in rye and barley activates the disease in susceptible individuals. Celiac sprue appears to be the only disease with a marked HLA-association in which the proteins that activate the disease currently are well known. However, bread wheat gliadins are a complex mixture of proteins that contain at least 40 different components. In the present study we have purified the major gliadin components of Scout 66 wheat and used these proteins to examine murine T cell proliferative responses to gliadin. Differences in T cell proliferation stimulated by alpha-, beta-, gamma-, and omega-gliadins paralleled the known structural differences among these proteins. After priming with whole gliadin, the components that stimulated T cell proliferation were the same as those recognized to activate celiac sprue in humans. Studies with reduced and alkylated A-gliadin (i.e., S-methyl A-gliadin) suggested that epitopes determined by the native conformation of A-gliadin may be important in its interaction with T cells. By using three different A-gliadin peptides that span the entire molecule, T cell proliferative responses were shown to be stimulated predominantly by antigenic determinants on the NH2-terminal peptide.