Peptide binding motifs for human major histocompatibility complex (MHC) class II (HLA) molecules of the DR subtype invariably predict a hydrophobic anchor residue near the N-terminus of the peptide (Rammensee et al. 1995). The crystal structure of HLA-DR1 (DRIll* 0101) complexed with the influenza haemagglutinin (HA) 307-319 peptide has revealed that the side chain of the major hydrophobic anchor residue, tyrosine at position 309, is bound in a deep and conserved hydrophobic pocket (Stern et al. 1994). This pocket is lined by DR~ residue 86, which can be either glycine (Gly) or valine (Val). Various studies have demonstrated that this Gly86/Va186 dimorphism at DRy86 can affect allorecognition (Lang et al. 1988; Johnson et al. 1991; de Koster et al. 1992; Demotz et al. 1993) and antigen presentation (Zeliszewski et al. 1990; Krieger et al. 1991; Busch et al. 1991; Ong et al. 1991). Moreover, this dimorphism has been shown to influence the stability of HLA-DR molecules in the detergent sodium dodecyl sulphate (SDS; Verreck et al. 1993). Together, these results suggest that different sets of peptides may be selected by Gly86/Valg6-DR variants. Indirect evidence for such differential peptide selection has been obtained by peptide binding studies (Krieger et al. 1991; Busch et al. 1991; Marshal et al. 1994) and the characterization of peptides associated to non-related Vala6-or GlyS6-containing DR allelic products (Vogt et al. 1994). In order to directly analyze the influence of DR1386 on peptide selection we performed a systematic analysis of the peptide content of Glys6/Va186 variants of the subspecificities DR1, DRll, DR13, and DR52 by pool sequencing as well as by the determination of individual peptide se-