The mechanisms by which glucocorticosteroids promote osteoclastogenesis in vitro are uncertain. As macrophage colony-stimulating factor (MCSF) is critical for osteoclastogenesis, we hypothesized that glucocorticosteroids might regulate membrane-bound MCSF (mMCSF) and soluble MCSF (sMCSF) production by stromal cells or osteoblasts. ST2 cells or murine calvarial osteoblasts (MOBs) were treated with dexamethasone (Dex; 100 nm) and/or 1,25-dihydroxyvitamin D[ 1,25(OH)₂D; 10 nm] for 3 days. Control values for mMCSF and sMCSF as units per 100,000 cells were 9 ± 1.4 and 511 ± 56 in ST2 cells and 5.9 ± 0.8 and 379 ± 47 in MOB cells, respectively. Dex increased mMCSF to 156 ± 16% and 143 ± 26% compared with the control value in ST2 and MOB cells, respectively, whereas 1,25-(OH)₂D caused increases of 195 ± 16% and 164 ± 21%. In the presence of both Dex and 1,25-(OH)₂D, mMCSF increased to 209 ± 24% and 216 ± 26% in the two cell types, respectively. 1,25-(OH)₂D caused modest increases in sMCSF, as expected, in both cell types (153 ± 6% and 122 ± 4%). Dex inhibited 1,25-(OH)₂D-stimulated sMCSF (115 ± 7% of control) in ST2 cells. Analysis of mMCSF transcript levels by semiquantitative RT-PCR revealed Dex-stimulated increases of 170 ± 11% in ST2 cells and 126 ± 16% in MOB cells compared with the control level. The increased expression of the transcript for sMCSF in the presence of Dex and 1,25-(OH)₂D, measured by both RT-PCR and Northern analysis (219 ± 53% and 242%, respectively), despite inhibition of sMCSF protein, indicated that the inhibitory effect of Dex in ST2 cells was posttranscriptional. Half-life studies showed that Dex prolonged MCSF messenger RNA from 2.8 to 7.5 h. These results suggest that Dex influences osteoclastogenesis by increasing the expression of mMCSF by accessory cells in culture.