The 25-hydroxyvitamin D3-24-hydroxylase mRNA is tightly and reciprocally regulated by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and parathyroid hormone (PTH). The upregulation of the 24-hydroxylase by 1,25(OH)2D3 is well established and occurs at the transcriptional level through two vitamin D response elements in the promoter of the gene. However, this induction is blocked by the protein synthesis inhibitor cycloheximide (CHX) indicating a protein component in the regulation pathway. CHX treatment reduced total vitamin D receptor (VDR) protein levels in cells, but reintroduction of VDR and/or retinoid X receptor protein into cells by transfection did not reduce the inhibition by CHX. This indicates that production of another transcription factor or mRNA-stabilizing protein synthesized in response to 1,25(OH)2D3 is required for optimal accumulation of 24-hydroxylase mRNA. PTH downregulates the 24-hydroxylase mRNA by affecting its stability. The half-life of 24-hydroxylase mRNA is reduced 4.2-fold in AOK-B50 cells by PTH. Untranslated regions of the 24-hydroxylase mRNA in reporter gene assays did not confer PTH responsiveness. Further analysis of the coding region of the rat 24-hydroxylase may reveal sites of action of PTH. J. Cell. Biochem. 88: 234–237, 2003. © 2002 Wiley-Liss, Inc.