There is a burgeoning literature on the contrasting role of intratumoral dendritic cells (DCs) and tumor-associated macrophages, making reliable identification of both cell types in clinical and experimental tissue sections important. However, because these cell types are closely related and share several differentiation antigens, their absolute distinction in tissue sections is difficult. We differentiated DCs and macrophages from monocytes in vitro, prepared cytospins and paraffin-embedded sections of the various cell populations, and tested a variety of antibodies that purportedly recognize monocytes and DCs for their capacity to react and distinguish cells after conventional formalin fixation. Cultured DCs but not macrophages were detected by fascin, DC-LAMP, and CD83 with a predictable increase in staining that paralleled their maturation. Staining by CD1a was found on immature DCs but was weak and absent on mature DCs and macrophages, respectively. CD14 and CD163 were characteristic for macrophages and absent on DCs. CD68, HLA-DR, and S100 did not discriminate between DCs and macrophages. We conclude that antigens such as HLA-DR and S100 are not in themselves sufficient for identification of DCs in formalin-fixed tissue sections, but that additional macrophage-specific (CD14, CD163) and DC-specific (CD1a, CD83, fascin, DC-LAMP) antigens should be used to distinguish cell types from each other and to provide information on their state of maturation.