Monocytic-like cell lines (MCLCs), including THP-1 and U-937 cells, are used routinely as surrogates for isolated human peripheral blood monocytes (PBMCs). In recent years these cell lines have been characterised based on mRNA expression levels of ion channels, G protein-coupled receptors1 (GPCRs) and inflammatory mediators2. To systematically evaluate MCLCs and PBMCs as model systems in which to study inflammation relevant to the pathogenesis of type II diabetes, we compared (i) mRNA expression for a panel of inflammation-relevant genes; (ii) cluster of differentiation (CD) marker expression and (iii) chemotactic responses of THP-1, U-937 and HL-60 MCLCs (with or without PMA treatment) and freshly-isolated human PBMCs, M1 macrophages and M1 macrophages activated with IFNγ/LPS. Messenger RNA expression analysis suggested that most genes were present at similar levels across all undifferentiated cells. Notably, IDO1, which encodes for indoleamine 2,3-dioxygenase and catabolises tryptophan to kynureninase (shown to be elevated in serum from diabetic patients3), was not expressed in any MCLC, but was present in PBMCs. There was little overall difference in the pattern of expression of CD markers across all cells, though absolute expression levels varied considerably. Functionally, THP-1 and PBMCs both migrated in response to chemoattractants in a transwell assay, with varying sensitivity to MCP-1, MIP-1α and LTB-4, in line with expression for their requisite target receptors. However, despite similar gene and CD expression profiles, U-937 cells were functionally impaired as no migration was observed to any chemoattractant. Our analysis reveals that MCLCs only partly replicate the genotypic and phenotypic properties of human PBMCs, but also that they exhibit substantial differences to each other, highlighting the necessity for careful interpretation of data generated using these immortalized cell lines, as previously recommended for PBMCs4.