COMPELLING evidence indicates that T cells recognize complexes formed by major histocompatibility complex-encoded molecules and antigenic peptide fragments^1–3. This is based largely on the ability of small synthetic peptides to substitute for naturally processed antigen in stimulating T cells. Naturally processed fragments of exogenous antigen are thought to arise by limited pro-teolytic degradation of native antigen inside acidic compartments of antigen-presenting cells^3–6, but until now no physiologically processed antigen has been directly analysed. Here we report the characterization of physiologically processed antigen eluted from mouse class II major histocompatibility complex I–E^d molecules. The antigenic material corresponds to a previously described antigenic determinant of hen egg lysozyme (HEL 107–116) and has a relative molecular mass M_r of about 2,000. HPLC analysis identified at least two or three separate molecular species, suggesting limited, albeit significant, heterogeneity of naturally processed peptides. Finally, under our experimental conditions, it was calculated that a substantial proportion (10–40%) of I–E^d molecules were occupied by these HEL-derived antigenic determinants.