Helper T lymphocytes recognize peptide antigens stably associated with class II major histocompatibility complex (MHC) glycoproteins on the surface of antigen-presenting cells and serve to regulate a wide variety of immune responses. A previous study from our laboratory had demonstrated that the functional association of various peptide antigens with the antigen-presenting cell membrane was increased at pH 5 as compared to pH 7, consistent with the potential role of acidic endosomal compartments in antigen processing. The mechanism for this effect was not determined. In the present study, assays using purified class II glycoprotein were used to further define this mechanism. The potential requirement for pH-dependent interactions involving non-MHC membrane components was excluded in functional assays with purified class II reconstituted in artificial membranes containing only neutral phospholipids and cholesterol. The association of HEL(104-120) with I-Ed, and OVA(323-339) with I-Ad, was increased at pH 5, as measured by activation of specific T cell hybridomas. An enzyme immunoassay was developed to measure the binding of biotin-labeled peptides to purified class II in detergent micelles. The pH dependence of binding paralleled our previous functional results. Optimum binding of biotin-HEL(104-120) to I-Ed was observed at pH approximately 4.5, whereas maximum binding of biotin-Myo(106-118) to I-Ad occurred at pH approximately 5.5. The latter peptide also bound weakly to I-Ed, but with a pH dependence similar to that observed using HEL(104-120). Further experiments with biotin-HEL(104-120)/I-Ed indicated that both the apparent affinity and the apparent concentration of peptide-binding sites are increased as hydrogen ion concentration is increased from pH 7 to pH 5. The effect of pH in this range was largely reversible and was not associated with a change in peptide dissociation that could be measured with our assay system. Binding was not inhibited in the presence of 1.5 M NaCl, suggesting that electrostatic interactions between HEL(104-120) and I-Ed are not essential for binding. It is proposed that protonation of a critical group(s) in the class II molecule regulates its capacity to form stable complexes with peptide. However, this effect alone does not fully account for the rapid kinetics of peptide binding observed in experiments with intact antigen-presenting cells.