The binding of immunogenic peptides to class II major histocompatibility molecules was examined at various pH values. We studied binding of peptides containing residues 52-61 from hen egg lysozyme (HEL) to I-Ak on fixed peritoneal macrophages or to solubilized affinity-purified I-Ak. Optimum binding occurred at pH 5.5-6.0 with accelerated kinetics relative to pH 7.4; equilibrium binding was also higher at pH 5.54.0 than at 7. Antigen processing involves the intracellular catabolism of proteins to produce immunogenic peptides that bind to class II major histocompatibility complex (MHC) molecules. A significant proportion of class II MHC molecules resides in endosomes (1-3), where binding of peptides appears to occur (2). Antigen processing involves late endosomes and lysosomes, since it is blocked at 180C (4). Efficient processing occurs with liposome-encapsulated antigens that are sequestered until delivery and release into lysosomes (5). These data suggest that immunogenic peptides produced in lysosomes may be recycled to endosomes to meet and bind to class II MHC molecules. Endosomes maintain an acidic luminal pH (about pH 6 for early endosomes and pH 5-5.5 in later endosomes, whereas lysosomes may attain pH 4.6-5.0). Alkalinization of the endosomal environment (e.g., by lysosomotropic amines or ionophores) disrupts endosomal/lysosomal processes (6), including antigen processing (7). In this context, class II MHC molecules would be expected to bind peptides efficiently at endosomal pH. Previously published data tend to support this hypothesis (8), but the effects of pH on peptide binding remain unclear in many aspects. Peptide dissociation has also been reported to be greater at acidic pH (pH 4.6-5.6) than at neutral pH (9), although other reports indicate that peptides remain stably bound in this pH range (10, 11).We have studied (12-14) the binding of peptides to the murine class II MHC molecule I-Ak on whole cells and as a solubilized afflinity-purified molecule (12-14 Immunogenic peptides were added in unbuffered normal saline and incubated at 37°C for 2 hr (peptides were synthesized and purified as described) (19). The cells were then washed and T-hybridoma cells were added in normal medium. T-cell responses (interleukin 2 secretion) were measured using a CTLL proliferation assay as described (18). We used the following T-cell hybridomas: 3A9, specific for the HEL peptide (21). For peptide dissociation experiments, the peptides were allowed to bind to fixed macrophages in normal medium. The cells were then washed, incubated at various pH values in the above buffers, and washed in regular medium prior to addition of T cells. For studies of the carbohydrates, fixed adherent macrophages or TA3 B-lymphoma cells were incubated with peptides in the presence of the carbohydrates, washed, and then incubated with T-hybridoma cells.