Macrophage colony stimulating factor (MCSF) is important for formation of osteoclasts. We investigated the ability of 1,25(OH)₂D₃ to regulate osteoblast production of MCSF. Mouse calvarial osteoblasts were cultured for 2 days ± 1,25(OH)₂D₃. Since 1,25(OH)₂D₃ decreased osteoblast proliferation by 17.6 ± 1% at 10 nM and 11 ± 4% at 1 nM, the effect of growth rate on MCSF secretion was examined. Limiting cell proliferation by serum did not affect MCSF production. 1,25(OH)₂D₃ (1 nM) increased MCSF production (U/10⁵ cells) maximally by 68 ± 33% (n = 3) with an ED₅₀ for 1,25(OH)₂D₃ of 5 × 10⁻¹¹ M. To investigate effects of 1,25(OH)₂D₃ on MCSF gene regulation, RT-PCR primers were designed to identify the mRNA coding for the membrane-bound isoform of MCSF. Simultaneous RT-PCR of glyceraldehyde-phosphate dehydrogenase (GAP) allowed semiquantitative assessment of MCSF mRNA between treatment groups expressed as the MCSF/GAP RT-PCR product ratio; both MCSF and GAP (+) primers were labeled with ³²P-ATP for phosphorimage quantitation. The membrane-bound MCSF/GAP PCR product ratio was not affected by proliferative rate when growth was limited by [serum]. The MCSF/GAP RT-PCR product ratio was dose dependently increased by 1,25(OH)₂D₃, maximally at 1 nM at 2.2 ± 0.2 = fold (n = 10). 1,25 (OH)₂D₃ also increased the expression of an RT-PCR MCSF/GAP product ratio which represented the secreted isoform of MCSF. The ability of 1,25(OH)₂D₃ to pretranslationally regulate expression of membrane-bound osteoblast MCSF may be important in osteoblast:osteoclast interactions.