Several studies have shown that the 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) receptor protein levels increase in response to 1,25-(OH)2D3. We have studied the mechanism of this regulation in both mouse fibroblasts and rat intestinal epithelial cells. Cell extracts and total RNA were prepared at varying times after addition of 1,25-(OH)2D3. The 1,25-(OH)2D3 receptor protein levels, measured using an immunoradiometric assay, rose significantly 2-3 h posttreatment and had risen 3-fold at 8 h. Concurrently, the 1,25-(OH)2D3 receptor mRNA content, measured using a ribonuclease protection assay, was not altered by 1,25-(OH)2D3 during this time. In cycloheximide-blocked cells, the administration of 1,25-(OH)2D3 markedly reduced the degradation rate of previously formed receptor. The 1,25-(OH)2D3 receptor protein half-life was determined as 4 h in the absence of 1,25-(OH)2D3 and increased to at least 8 h in the presence of 1,25-(OH)2D3. We also measured the 1,25-(OH)2D3 receptor mRNA levels in the duodena and kidney of vitamin D-deficient rats after a single 150-pmol injection of 1,25-(OH)2D3. Again, we found that 1,25-(OH)2D3 receptor mRNA levels were not changed in these tissues after 1,25-(OH)2D3 treatment. Therefore, the elevation of the 1,25-(OH)2D3 receptor protein following 1,25-(OH)2D3 administration is apparently the result of increased receptor protein lifetime and not increased transcription.