The presence of macrophages in dental pulp is well known. However, whether these macrophages proliferate and differentiate in the dental pulp in situ, or whether they constantly migrate from the blood stream into the dental pulp remains unknown. We have examined and compared the development of dental pulp macrophages in an organ culture system with in vivo tooth organs to clarify the developmental mechanism of these macrophages. The first mandibular molar tooth organs from ICR mice aged between 16 days of gestation (E16) to 5 days postnatally were used for in vivo experiments. Those from E16 were cultured for up to 14 days with or without 10% fetal bovine serum. Dental pulp tissues were analyzed with immunohistochemistry to detect the macrophages and with reverse transcription and the polymerase chain reaction (RT-PCR) for the detection of factors related to macrophage development. The growth curves for the in vivo and in vitro cultured cells revealed similar numbers of F4/80-positive macrophages in the dental pulp. RT-PCR analysis indicated the constant expression of myeloid colony-stimulating factor (M-CSF) in both in-vivo-and in-vitro-cultured dental pulp tissues. Anti-M-CSF antibodies significantly inhibited the increase in the number of macrophages in the dental pulp. These results suggest that (1) most of the dental pulp macrophages proliferate and differentiate in the dental pulp without a supply of precursor cells from the blood stream, (2) M-CSF might be a candidate molecule for dental pulp macrophage development, and (3) serum factors might not directly affect the development of macrophages.