Thaw-mount autoradiographic studies after injection of 3H-1,25-D3 were conducted on 18- and 20-day-old rat fetuses. In maxillary bones, ribs, and tibia, nuclear concentration of radioactivity was found in osteoprogenitor cells and osteoblasts. Osteocytes and chondrocytes in epiphyseal plates were either unlabeled or weakly labeled. In competition experiments, nuclear concentration of radioactivity was blocked by the injection of a high dose of nonradioactive 1,25-D3 prior to the administration of the labeled hormone, but not by a similar dose of nonradioactive 25-D3. The results are interpreted as indicating that osteoprogenitor cells and osteoblasts are target cells for the direct action of 1,25-D3 on fetal bone.