The T cell-independent antibody response to polysaccharide antigen (Ag) is believed to result from their inability to bind major histocompatibility complex (MHC) restriction elements. However, recent studies using glycosylated analogues of known immunogenic peptides revealed that glycopeptides can interact with MHC molecules and are able to elicit specific T cell responses in experimental animals. This raises questions about the possible role which carbohydrates can play in T cell responses following natural exposure to glycoprotein antigens. Analyzing the fine specificity of the human T cell response against the major bee venom allergen phospholipase A2 (PLA), a 16-20-kDa protein glycosylated at a single site (Asn13), we have identified several T cell clones which proliferate in response to the glycoprotein but not to its non-glycosylated variants. Neither the carbohydrate moiety alone nor the combination of carbohydrate and non-glycosylated protein could substitute for the intact glycoprotein. Antibody directed against the carbohydrate moiety inhibited Ag-induced proliferation of these clones whereas control clones with known peptide specificity were not affected, providing additional evidence for the involvement of carbohydrates in T cell recognition. Moreover, peripheral blood mononuclear cells of two individuals from whom glycosylation-dependent T cell clones have been isolated showed significantly higher proliferation in response to glycosylated compared to non-glycosylated Ag, suggesting that glycosylation can contribute in some cases extensively to the immunogenicity of a glycoprotein Ag. Thus, this report shows that glycosylation-dependent Ag recognition by T cells can also occur following natural exposure to a glycoprotein.