Familial isolated hypoparathyroidism (FIH) is an inherited metabolic disorder characterized by hypocalcemia and hyperphosphatemia due to deficient secretion of biologically active PTH. We used denaturing gradient gel electrophoresis to screen for mutations in exon 1 and the coding region of the preproPTH gene. Exons 1, 2, and 3 of the preproPTH gene and flanking intronic regions were amplified by polymerase chain reaction using primers that were designed employing the MELT-MAP program. One oligonucleotide from each primer pair was synthesized with a 5'-GC-clamp. Screening of amplified DNA from normal subjects and patients with FIH revealed two single base changes that altered migration of amplified preproPTH gene fragments through denaturing gels: 1) an A----G transition in intron 1; 10 nucleotides upstream of exon 2; and 2) a C----A transversion in exon 3 that conserves the arginine residue at codon 52 (CGA----AGA). By contrast, we did not detect pathogenic mutations in amplified regions of the preproPTH genes of 18 affected members of 5 FIH kindreds. The two polymorphisms occur frequently, and were therefore used to perform linkage analysis in 5 multiplex FIH families. Linkage analysis was inconclusive in 2 families and showed discordance between hypoparathyroidism and any of the preproPTH gene alleles in 2 other families. In another family, analysis was suggestive of linkage between hypoparathyroidism and inheritance of a specific preproPTH gene allele. These results indicate that denaturing gradient gel electrophoresis can be used to identify mutations in defined regions of the preproPTH gene, and to examine linkage of specific preproPTH alleles and inherited disorders of mineral metabolism.