In cultured 19 day fetal rat calvaria, osteoclasts first appear after 48 h, more rapidly than with other cultured embryonic long bone rudiments. This may be because the calvarial osteoclast precursors are more differentiated or intramembraneous bone is a more powerful stimulus for osteoclast maturation than endochondral bone. To investigate this further, 19 day calvariae were stripped of their endocranial membranes, devoiding them of osteoclast precursors, and cocultured with the membranes or with other sources of these cells, such as bone marrow, fetal liver, spleen, and blood. There was similar recruitment of mature osteoclasts onto the surface of the "stripped" calvariae from the endocranial membranes and from the hematopoietic tissues after 48 h culture. Intact 19 day fetal calvariae were cultured with human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) or with 1,25-dihydroxyvitamin D3, [1,25-(OH)2D3], each thought to influence different stages of osteoclast maturation. They stimulated osteoclast recruitment, although 1,25-(OH)2D3 was effective only in the first 24 h of culture. They also increased osteoclast recruitment from fetal liver onto stripped calvariae. When intact bones were cultured with hrGM-CSF and 1,25-(OH)2D3 together, osteoclast number decreased but their area increased. Calvariae therefore appear to contain osteoclast precursors at earlier (GM-CSF-sensitive) and later [1,25-(OH)2D3-sensitive] stages. As recruitment onto stripped calvariae was similar whichever source of precursors was used, it is likely that calvarial bone matrix is an important influence on rapid osteoclast maturation in these bones in vitro.