Osteoblastic cell cultures from fetal rat calvariae have provided a popular model for studying the effects of dexamethasone (DEX) and 1,25 dihydroxyvitamin D-3 [1,25(OH)(2)D-3] on gene expression but data from murine calvarial cells are scarce. Species-specific responses of rat and mouse osteoblastic cells to these hormones have been reported previously. In the present study, we investigated the effects of DEX and 1,25(OH)(2)D-3 on expression of the osteoblastic phenotype by mouse calvarial cells. These murine osteoblast-like (MOB) cells expressed alkaline phosphatase (ALP) activity and osteocalcin and formed calcified nodules. Unlike the rat calvarial cells, ALP activities and nodule formation in MOB were inhibited by DEX. 1,25(OH)(2)D-3 enhanced and DEX lowered the amount of osteocalcin synthesized by MOB. 1,25(OH)(2)D-3 did not affect the number of nodules, but increased their sizes. Treating the cells for 2 days with only DEX at the beginning of the culture enhanced the effect of 1,25(OH)(2)D-3 on ALP. We found that in murine calvarial cells, DEX inhibits and 1,25(OH)(2)D-3 enhances ALP activity, osteocalcin synthesis, and calcified nodule formation. This is in contrast to previous reports of rat calvarial cells where DEX is a positive and 1,25(OH)(2)D-3 can be a negative regulator of the osteoblastic phenotype. These results suggest that profound species-specific differences exist between mice and rats in the regulation of the osteoblastic phenotype.