Fibroblasts from three patients with vitamin D-dependency rickets type II were used to study mutations in the 1,25-dihydroxyvitamin D3 receptor responsible for this hereditary disease. Normal human fibroblasts contain 43 +/- 13 fmol receptor/mg protein as determined by immunoradiometric assay and 22 +/- 3 fmol/mg by ligand binding assay. The fibroblasts from the rachitic patients contained no receptor detectable by either method. The 1,25-(OH)2D receptor cDNA for cells from each kindred was produced from total RNA using reverse transcription and polymerase chain reaction amplification. When these cDNAs were sequenced, it was found that each cell line contained a nucleotide substitution resulting in a stop codon in the coding sequence. The predicted resultant receptor protein is 69 amino acids long in one family, and 148 and 291 amino acids long in two other families. These truncated proteins have little or no 1,25-dihydroxyvitamin D3-binding domain accounting for 1,25-dihydroxyvitamin D resistance.