Although studies have been performed to characterize responses of macrophages from individual anatomical sites (e.g., alveolar macrophages) or of murine-derived macrophage cell lines to microbial ligands, few studies compare these cell types in terms of phenotype and function. We directly compared the expression of cell surface markers and functional responses of primary cultures of three commonly used cells of monocyte-macrophage lineage (splenic macrophages, bone marrow-derived macrophages, and bone marrow-derived dendritic cells) with those of the murine-leukemic monocyte-macrophage cell line, RAW 264.7. We hypothesized that RAW 264.7 cells and primary bone marrow-derived macrophages would be similar in phenotype and would respond similarly to microbial ligands that bind to either Toll-like receptors 2, 3, and 4. Results indicate that RAW 264.7 cells most closely mimic bone marrow-derived macrophages in terms of cell surface receptors and response to microbial ligands that initiate cellular activation via Toll-like receptors 3 and 4. However, caution must be applied when extrapolating findings obtained with RAW 264.7 cells to those of other primary macrophage-lineage cells, primarily because phenotype and function of the former cells may change with continuous culture.