It is well known that T cells recognize antigen as processed peptides bound to major histocompatibility complex molecules on the surface of antigen-presenting cells. Recently, it has been shown that T cells can specifically recognize synthetic glycopeptides. However, whether glycopeptides are selected for presentation during antigen processing of glycoproteins and eventually elicit carbohydrate-specific T cells is still an open question. In this study, we utilized synthetic glycopeptides to analyze T cell recognition of the naturally glycosylated immunodominant peptide representing type II collagen (CII) residues 256-270. In this peptide, lysines at positions 264 and 270 may be post-translationally modified by hydroxylation and subsequent O-linked glycosylation with beta-galactosyl or alpha-glucosyl-(1-->2)-beta-galactosyl residues. T cell hybridomas established from type II collagen-immunized mice specifically recognized CII 256-270 with either galactose or glucosyl-galactose at position 264. The T cell hybridoma recognizing glucosyl-galactose displayed no cross-reactivity either to galactose or to the structurally different alpha-galactosyl-(1-->4)-beta-galactose. Furthermore, the T cell hybridoma recognizing galactose did not cross-react to glucosyl-galactose or galactosyl-galactose, indicating that the antigen-presenting cells (bulk spleen cells, lipopolysaccharide-stimulated spleen cells, anti-CD40-stimulated spleen cells, peritoneal exudate cells or CFA-primed lymph node cells) inefficiently processed carbohydrates when the antigen was given as a glycopeptide.