To study the processing and presentation of endogenously synthesized Ag to class II MHC-restricted T cells, hen egg lysozyme (HEL), either tagged with a peptide that confers retention in the endoplasmic reticulum (HEL.KDEL), or in the secretory form (HELs), was stably expressed in LK-35.2 B hybridoma cells. Presentation of HEL peptides bound to class II molecules was assessed by activation of specific T cell hybridomas recognizing seven different epitopes derived from exogenous HEL. The presentation of endogenously synthesized HEL was not caused by reuptake of secreted of shed Ag. All the HEL epitopes examined were efficiently presented after processing of endogenous HEL by HELs-transfected LK-35.2 cells. Processing of HEL tagged with KDEL, however, gave rise to presentation of only six of the seven HEL epitopes. The epitope included in the HEL sequence 112-124 was not presented by HEL.KDEL-transfected B cells. In addition, two of the four T cell hybridomas recognizing HEL 116-129 together with I-Ak molecules were not activated by HEL.KDEL, and three other epitopes were presented with lower efficiency as compared with HELs. Thus, endogenously synthesized HEL in secretory form gives rise to a set of class II-binding epitopes indistinguishable from exogenous HEL, whereas endoplasmic reticulum-retained HEL generates a similar but not identical set of epitopes. The endosomal protease inhibitor leupeptin prevented presentation of the epitope 108-116, but not 46-61, both by HELs and HEL.KDEL transfected cells, indicating a requirement for endosomal processing in both cases. In addition, the presentation of peptides derived from endogenously synthesized, either secretory or endoplasmic reticulum-retained HEL, could be inhibited by lysosomotropic amines, further indicating that the intracellular route of class II molecules presenting peptides derived from endogenous Ag intersects the acidic endosomal compartment.