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While they are well studied in animal models, it is still strongly debated what specifies most accurately the phenotype and functioning of microglia in the human brain. In this study, we therefore isolated microglia from postmortem human brain tissue of corpus callosum (CC) and frontal cortex (CTX). The cells were phenotyped for a panel of typical microglia markers and genes involved in myeloid cell biology. Furthermore, their response to pro\u2010 and anti\u2010inflammatory stimuli was assessed. The microglia were compared to key human myeloid cell subsets, including monocytes, monocyte\u2010derived macrophages and monocyte\u2010derived dendritic cells, and several commonly used microglial cell models. Protein and mRNA expression profiles partly differed between microglia isolated from CC and frontal cortex and were clearly distinct from other myeloid subsets. Microglia responded to both pro\u2010 (LPS or poly I:C) and anti\u2010inflammatory (IL\u20104 or dexamethasone) stimuli. Interestingly, pro\u2010inflammatory responses differed between microglia and monocyte\u2010derived macrophages, as the former responded more strongly to poly I:C and the latter more strongly to LPS. Furthermore, we defined a large phenotypic discrepancy between primary human microglia and currently used microglial cell models and cell lines. In conclusion, we further delineated the unique and specific features that discriminate human microglia from other myeloid subsets, and we show that currently used cellular models only partly reflect the phenotype of primary human microglia. 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