Antigen-specific CD4⁺ T cells are implicated in the autoimmune disease systemic lupus erythematosus (SLE), but little is known about the peptide antigens that they recognize and their precise function in disease. We generated a series of MHC class II tetramers of I-E^k–containing peptides from the spliceosomal protein U1-70 that specifically stain distinct CD4⁺ T-cell populations in MRL/lpr mice. The T-cell populations recognize an epitope differing only by the presence or absence of a single phosphate residue at position serine¹⁴⁰. The frequency of CD4⁺ T cells specific for U1-70(131-150):I-E^k (without phosphorylation) correlates with disease severity and anti–U1-70 autoantibody production. These T cells also express RORγt and produce IL-17A. Furthermore, the U1-70–specific CD4⁺ T cells that produce IL-17A are detected in a subset of patients with SLE and are significantly increased in patients with mixed connective tissue disease. These studies provide tools for studying antigen-specific CD4⁺ T cells in lupus, and demonstrate an antigen-specific source of IL-17A in autoimmune disease.