The effects of granulocyte-macrophage colony stimulating factor (GM-CSF), ntacrophage colony stimulating factor (M-CSP), and interleukin 3 (IL3j on osteoclast formation were tested by incubation of murine hemopoietic cells on plastic coverslips and bone slices with CM-CSF, M-CSF, or IL3, with or without 1,25(OH), vitamin D, (1,2S(OH),D,). Osteoclastic differentiation was detected after incubation by scanning electron microscopical examination of bone slices for evidence of osteoclastic excavations, and by autoradiographic assessment of cells for 1,25(0H),D,-calcitonin (C-I) binding. The differentiation of CT-receptor-positive cells preceded bone resorption, but the number that developed correlated with the extent of bone resorption (r = 0.88). M-CSF and GM-CSF substantially reduced bone resorption and CT-receptor-positive cell formation. The degree of inhibition of bone resorption could not be attributed to effects on the function of mature cells, since M-CSF inhibits resorption by such cells only by 5O%, and GM-CSF has no effect. GM-CSF inhibited the development of mature function (bone resorption) to a greater extent than it inhibited CT-receptor-positive cell formation. Since CT-receptor expression antedated resorptive function, this suggests that LM-CSF resulted in the formation ot reduced numbers of relatively immature osteoclasts. This suggests that it may exert a restraining effect on the maturation of cells undergoing osteoclastic differentiation in response to 1,25(C)Hj,O,. Conversely, IL3, which also has no effect on mature osteuclasts, by itself induced CT-receptor expression but not bone resorption; in combination with 1,2S(OH),D, it induced a threefold increase in bone resorption and CTreceptor-positive cells compared with cultures incubated with 1,2S(OH),D, alone. IL3 did not induce CT-receptors in peritoneal macrophages, blood monocytes, or J 774 cells. The results suggest that IL3 induces only partial maturation