Macrophages exert a crucial, but still incompletely known, role in complex disorders such inflammatory, immunological, and infectious diseases. A differential proteomic approach should help to elucidate the macrophage dysfunctions involved in these diseases. With this goal in mind, we established the first two-dimensional maps of the human macrophage proteome and secretome. Intracellular and secreted proteins were extracted from monocyte-derived macrophages obtained from healthy donors (n = 16), and separated by two-dimensional gel electrophoresis. Silver-stained gels were analyzed using Progenesis software. A high level of between-gel reproducibility was obtained, allowing us to generate two patterns specific of the macrophage proteome and secretome, respectively. A total of 127 and 66 distinct intracellular and secreted polypeptide spots, corresponding to 100 and 38 different proteins, respectively, were identified by matrix assisted laser desorption/ionisation-mass spectrometry. The two-dimensional reference maps and databases resulting from this study confirm that macrophages are involved in a wide range of biological functions, and that they provide a useful tool for a wide array of investigators involved in macrophage biology, allowing to investigate the macrophage protein changes associated with various disorders or environmental stimuli.