Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced bone marrow-derived cells (BMCs) and primary peritoneal exudate cells (PECs) are usually used for antigen presentation in in vitro experiments. In order to expound their tendency for uptake and antigen presentation, we compared differences in the degree of phagocytosis, the expression of co-stimulatory molecules, and the activation of T lymphocytes between these two cell types. These assays used the F4/80 marker expression, as it is the general marker for macrophages. The BMC population was found to contain both F4/80(bright) and F4/80(dim) subtypes, while PECs were mainly composed of the F4/80(bright) subtype. Expression levels of cell surface co-stimulatory molecules, CD80, CD86, CD54, and CD40, were significantly higher for F4/80(+)BMCs than F4/80(+)PECs. Their expressions were further upregulated for F4/80(+)BMCs than for F4/80(+)PECs after stimulation with flagellin. F4/80(+)BMCs had a weaker ability to phagocytize microbeads than F4/80(+)PECs (P < 0.05), and we determined no relationship between F4/80 expression and phagocytosis. T lymphocytes were activated more efficiently after incubation with BMCs pulsed with flagellin than with pulsed PECs. In this study, F4/80(+)BMCs and F4/80(+)PECs represent the bone marrow-derived macrophages (BMMs) and peritoneal macrophages (PMs), respectively. These results indicate that PMs showed greater potential for phagocytosis, whereas GM-CSF-induced BMMs showed a tendency toward antigen presentation.