DRB1*08:01 (DR0801) and DRB1*11:01 (DR1101) are highly homologous alleles that have opposing effects on susceptibility to primary biliary cirrhosis (PBC). DR0801 confers risk and shares a key feature with other HLA class II alleles that predispose to autoimmunity: a nonaspartic acid at beta57. DR1101 is associated with protection from PBC, and its sequence includes an aspartic acid at beta57. To elucidate a mechanism for the opposing effects of these HLA alleles on PBC susceptibility, we compared the features of epitopes presented by DR0801 and DR1101. First, we identified DR0801- and DR1101-restricted epitopes within multiple viral Ags, observing both shared and distinct epitopes. Because DR0801 is not well characterized, we deduced its motif by measuring binding affinities for a library of peptides, confirming its key features through structural modeling. DR0801 was distinct from DR1101 in its ability to accommodate charged residues within all but one of its binding pockets. In particular, DR0801 strongly preferred acidic residues in pocket 9. These findings were used to identify potentially antigenic sequences within PDC-E2 (an important hepatic autoantigen) that contain a DR0801 motif. Four peptides bound to DR0801 with reasonable affinity, but only one of these bound to DR1101. Three peptides, PDC-E2145-159, PDC-E2(249-263), and PDC-E2(629-643), elicited high-affinity T cell responses in DR0801 subjects, implicating these as likely autoreactive specificities. Therefore, the unique molecular features of DR0801 may lead to the selection of a distinct T cell repertoire that contributes to breakdown of self-tolerance in primary biliary cirrhosis, whereas those of DR1101 promote tolerance.