Monocyte/macrophages are professional antigen-presenting cells of the cellular immune system, serving to generate peptides for major histocompatibility complex (MHC) class II-restricted recognition by CD4+ T-lymphocyte effector cells. Antigen presentation by these cells involves the internalization of extracellular proteins and their fragmentation within vacuolar compartments. The resulting peptides become associated with MHC class II molecules. The final destination of exogenous peptide antigens, however, is not absolute in monocytes. Processed peptides, derived from exogenous proteins, can also associate with MHC class I molecules. To study simultaneous presentation of peptides derived from exogenous and endogenous proteins by human leucocyte antigen (HLA) class I molecules, we isolated the peptides from a human immunodeficiency virus nef transfected U937 monocytic cell line. The HLA class I-bound peptides were separated by reverse phase-high performance liquid chromatography. Comparison of the peptide sequence data with protein databases revealed that the peptides derived from extracellular, as well as intracellular, proteins, suggesting that monocytes have a more generalized MHC class I antigen-processing pathway than previously documented.