We studied factors affecting major histocompatibility complex class II (MHC-II)-restricted presentation of exogenous peptides at the surface of macrophages. We have previously shown that peptide presentation is modulated by surface-associated proteolytic enzymes, and in this report the role of the binding of MHC-II molecules in preventing proteolysis of exogenous synthetic peptides was addressed. Two peptides containing CD4 T-cell epitopes were incubated with fixed macrophages expressing binding and non-binding MHC-II, and supernatants were analysed by high-performance liquid chromatography and mass spectrometry to monitor peptide degradation. The proportion of full-length peptides that were degraded and the number of peptide fragments increased when non-binding macrophages were used, leading to reduction in peptide presentation. When MHC-II molecules expressed on the surface of fixed macrophages were blocked with monoclonal antibody and incubated with peptides and the supernatants were transferred to fixed macrophages, a significant reduction in peptide presentation was observed. Peptide presentation was up-regulated at pH 5.5 compared to neutral pH, and the latter was found to be the pH optimum of the proteolytic activity of the surface enzymes involved in the degradation of exogenous peptides and proteins. The data suggest that MHC-II alleles that bind peptides protect them from degradation at the antigen-presenting cell surface for presentation to CD4 T cells and we argue that this mechanism could be particularly pronounced at sites of inflammation.