Th cells recognize a processed form of Ag in association with class II histocompatibility molecules expressed on the surface of APC. The physical nature of the cell surface association of physiologically processed Ag was investigated by using membranes isolated from Ag-pulsed APC. Such membranes were sufficient to directly activate class II-restricted T cell hybridomas without further Ag processing. T cell-stimulating activity remained after treatment of membranes in harsh conditions, including pH 4.0, pH 9.0, high salt, and chaotropic solvents. Activity was lost after exposure to pH 2.0 or protease. The capacity of pH 2.0 (but not protease) treated membranes to present artificially processed, peptide Ag to T cells suggests that exposure to pH 2.0 results in the selective dissociation of processed Ag from membranes. Similar results were obtained in parallel experiments with peptide-pulsed membranes. No qualitative differences were found between physiologically processed Ag and peptide Ag with respect to their remarkably stable association with the APC plasma membrane.