The effect of cholecalciferol metabolites on ornithine decarboxylase activity and on DNA synthesis in developing long bones was investigated in vitamin D-depleted rats. In the epiphysis there was a 6.4-fold increase in ornithine decarboxylase activity 5 h after a single injection of 24R,25-dihydroxycholecalciferol but not of 24S,25-dihydroxycholecalciferol or other vitamin D metabolites. In comparison, in the diaphysis and duodenum, 1 alpha,25-dihydroxycholecalciferol, but not other vitamin D metabolites, caused a 3-3.5-fold increase in the enzyme activity. The enzyme activity in the tissues examined attained a maximal value at 5 h after the injection of the metabolites. The activity of ornithine decarboxylase in the epiphysial region increased dose-dependently as the result of a single injection of 24R,25-dihydroxycholecalciferol and attained a maximal value at a dose between 30 and 3000 ng. In addition, administration of 24R,25-dihydroxycholecalciferol, but not 24S,25-dihydroxycholecalciferol or other metabolites, caused within 24 h a 1.7-2.0-fold increase in [3H]thymidine incorporation into DNA of the epiphyses of tibial bones. In comparison, 1 alpha,25-dihydroxycholecalciferol caused a 1.5-fold increase in [3H]thymidine incorporation into DNA of the diaphyses and of the duodenum. The present data indicate that 24R,25-dihydroxycholecalciferol is involved in the regulation of epiphyseal growth, whereas 1 alpha,25,dihydroxycholecalciferol stimulates the proliferation of cells in the diaphysis of long bones and in the intestinal mucosa.