Progenitor cells for several mesenchymally derived cell types exist within freshly isolated fetal rat calvaria (RC) cell populations. We have characterized the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on the differentiation of adipocytes from primary RC cells and compared these effects with those of dexamethasone (Dex). RC cells were plated at 3 x 10(4)/35-mm dish, and cultures were maintained for 14-19 days in alpha-Minimum Essential Medium containing 10% fetal bovine serum, 50 micrograms/ml ascorbic acid, 10 mM Na beta-glycerophosphate, and 0.1-100 nM 1,25-(OH)2D3 or 1-1000 nM Dex. Morphological (quantitation of adipocyte foci number and area after staining cultures with Sudan IV) and biochemical (glycerol-3-phosphate dehydrogenase activity) methods of assessing adipogenesis were used. In the presence of 1,25-(OH)2D3, adipocyte foci developed about 3 days after confluency as clusters of rounded or stellate cells. Stimulation of adipocyte foci development was dose dependent from 0.1-100 nM and was maximal with 10 nM 1,25-(OH)2D3; half-maximal stimulation occurred at about 1 nM. The presence of ascorbic acid and beta-glycerophosphate was not required for 1,25-(OH)2D3-induced stimulation of adipocytes, but both significantly increased the number of adipocyte foci in the presence of 1,25-(OH)2D3. The critical period for initiation of adipocyte differentiation with 1,25-(OH)2D3 was between 1-9 days, and once committed along the adipogenic pathway, adipocytes maintained their differentiated state in the absence of 1,25-(OH)2D3. Short term (48-h) pulses of 1,25-(OH)2D3 resulted in slight, but significant, increases in adipocyte formation. Other vitamin D3 metabolites were less effective than 1,25-(OH)2D3 in stimulating adipocyte differentiation. Dex (1-100 nM) also caused a dose-dependent increase in the differentiation of adipocyte foci in RC cell cultures. The adipocyte foci that developed in the presence of Dex frequently appeared earlier in culture, i.e. when cells reached confluency on days 6-7, and were more diffuse than those forming with 1,25-(OH)2D3. The stimulation of adipocyte differentiation by 1,25-(OH)2D3, however, was greater than that by Dex in mixed RC II-V cells. The combined effects of 1,25-(OH)2D3 and Dex were additive at low concentrations and synergistic at higher concentrations of either 1,25-(OH)2D3 or Dex. The data show that bone cell populations isolated from fetal RC contain adipocyte progenitors and that 1,25-(OH)2D3 as well as Dex are potent regulators of adipocyte differentiation within these bone cell populations.