Mononuclear phagocytes (MP; dendritic cells, monocytes, tissue macrophages, and microglia) maintain tissue homeostasis and provide a first line of defense against invading pathogens. In specific circumstances, MPs also induce inflammatory responses and as such affect disease onset and progression. Despite intensive research into MP biology, little is known of the functional and molecular properties of individual MP subtypes. Using a novel proteomics platform, unique protein patterns and protein identities were observed among populations of spleen and bone marrow macrophages and microglia. Cells were obtained from C57BL/6 mice and were cultivated in macrophage colony-stimulating factor. MP subtypes were indistinguishable by morphological or antigenic criteria. Protein profiling by Surface Enhanced Laser Desorption Ionization-Time of Flight (SELDI-TOF) ProteinChip assays with weak cationic exchange chips showed unique MP spectral profiles. Corresponding protein fractions were recovered by high performance liquid chromatography and identified by liquid chromatography tandem mass spectrometry. The results provide a unique means to distinguish microglia from other MP subtypes.