Vitamin D metabolites are able to change plasma calcitonin (CT) levels, but nothing is known about a possible effect at the CT gene level. Here we have investigated the acute effects of 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2D 3 on the CT biosynthetic activity of thyroid glands from adult rats. Plasma CT levels were significantly increased (× 2) 1 and 2 h after 1,25-(OH) 2D 3 injection in the face of unchanged plasma calcium values. The thyroidal CT content also was unchanged. A 2-fold increase in CT mRNA level measured by dot-blot hybridization occurred 1 and 2 h after 1,25-(OH) 2D 3 administration. Expression of CT gene products was examined in the rabbit reticulocyte lysate cell-free translation assay. After polyacrylamide gel electrophoresis, specific immunoprecipitates were autoradiographed and quantified by integration. A single precursor of M r ≊ 15000 could be specifically immunoprecipitated with CT antisera. A 3–4-fold rise in translatable CT mRNA activity was observed 1 and 2 h after 1,25-(OH) 2D 3 injection. Thus, parallel changes in CT mRNA level, CT mRNA activity and plasma CT levels were observed in adult female rats after administration of 1,25-(OH) 2D 3. These findings demonstrate for the first time that 1,25-(OH) 2D 3 enhanced CT gene expression in the face of unchanged plasma calcium levels.