Rat osteoblasts were cultured aboard a space shuttle for 4 and 5 days. Cells were treated with 1 nM 1alpha,25-dihydroxyvitamin D(3) (VD) for the last 1 day. The conditioned media were harvested. Cells were solubilized with guanidine solution on board. We examined microgravity effects on the production/expression of osteocalcin, bone sialoprotein (BSP), and VD receptor (VD-R) in osteoblasts. Under VD treatment, the osteocalcin protein level was 243 +/- 117 and 1,718 +/- 534 pg/microg cellular DNA in flight cultures and ground controls, respectively. Without VD treatment, the osteocalcin protein level was not different between flight cultures and ground controls. The osteocalcin mRNA level in the VD-treated flight cultures was as low as 16% of that in ground controls. The VD-R mRNA level in the VD-treated flight cultures was also decreased to 16% of that in ground controls. Microgravity would suppress the VD-inducible production of osteocalcin but not the basal productivity. The BSP mRNA level was increased by microgravity. VD/VD-R binds to the vitamin D-responsive element (VDRE) on the target genes. The rat osteocalcin gene is positively regulated via "enhancer" VDRE, whereas the rat BSP gene is negatively regulated via "repressor " VDRE. Microgravity might modulate osteoblast responsiveness to VD through the suppression of VD-R.