We examined the specificity of glycopeptide-specific CD4 T cells following procedures similar to those previously reported by us. The disaccharide galabiose (Gal alpha 1-4Gal) was attached to the middle of the 52-61 peptide of hen egg lysozyme. This peptide is well known to bind to I-Ak molecules. CBA/J mice were immunized and T cell hybridomas were derived from the popliteal lymph node T cells. For this study, we selected hybridomas that recognized galabiose conjugated to 52-61 at residue Ser 56. We demonstrate here that these hybridomas showed specificity for galabiose and not cellobiose (Glc beta 1-4Glc). Peptides containing galabiose at residue 53 did not stimulate the T cell hybridomas and neither did galabiose conjugated to the 34-45 peptide of HEL. Acetylation of the hydroxyl groups of the disaccharide resulted in loss of T cell reactivity. These results need to be contrasted with those in which the T cells were directed to galabiose, attached to the amino terminus of 52-61 or to Ser at residue 53. With these results, the fine specificity of recognition of the disaccharide was not apparent. Our results indicate two sets of glycopeptide-specific T cells. One is probably induced by a conformational change induced by the disaccharide on the peptide bound to class II MHC molecules. The second set contains elements of specificity for both the disaccharide and the peptide.