Class II-associated invariant chain peptides (CLIPs) compete with natural allele-specific ligands for binding to several purified HLA-DR molecules. Truncation and substitution analysis showed that a minimal sequence of 13 amino acids is sufficient for excellent binding to DR17 and DR1. Hydrophobic residues at relative positions 1 and 9 (P1 and P9) which are shared among these DR-ligands, and are found to be anchored in complementary pockets by x-ray crystallography allow specific binding. Two flanking residues at either end next to the specific contact sites Met107 and Met115 contribute to binding irrespective of their side chains, suggesting H-bonds to the major histocompatibility complex (MHC) molecule. Thus, CLIPs behave like conventional ligands, however, lack their allele-specific contact sites. Introduction of the DR17-specific contact site aspartate at P4 dramatically improves invariant chain-peptide binding to DR17, but reduces DR1 binding. By contrast, binding to DR1, but not DR17 is strongly improved after introduction of the DR1-specific contact site alanine at P6. In addition, analyzing the fine specificity of the hydrophobic contact sites at P1 and P9, CLIP variants reflected the allele-specific preferences of DR17- or DR1-ligands, respectively, for aliphatic or aromatic residues. Alignment studies suggest that CLIPs are designed for promiscuous binding in the groove of many MHC class II molecules by taking advantage of one or more supermotifs. One such supermotif, for example, does not include the DR17-specific contact site aspartate at P4, which in conventional natural ligands like Apolipoprotein (2877-94) is necessary to confer a stable conformation. Introduction of aspartate at P4 generates a CLIP variant that is stable in the presence of sodium dodecyl sulfate, such as allele-specific ligands. Studying the stability of class II-CLIP complexes at pH 5, we found that CLIPs, similar to anchor-amputated ligands, can be released from class II molecules, in contrast to conventional natural ligands, which were irreversibly bound. Taken together, our data provide compelling evidence that CLIP peptides bind into the class II groove.