The rate-limiting, hormonally regulated step in the biological activation of vitamin D is the 1α-hydroxylation of 25OHD to 1,25(OH)2D in the proximal renal tubule. This reaction is catalyzed by the mitochondrial cytochrome P450 enzyme, P450c1α. We have recently cloned the P450c1α cDNA from human keratinocytes, demonstrated that it is the same gene as that expressed in the kidney, and used RT-PCR of keratinocyte mRNA from a patient with VDDR-1 to show that mutations in P450c1α cause this disease (Mol Endocrinol 11:1961, 1997). We then cloned and sequenced the unique-copy CYP40 gene on chromosome 12 that encodes P450c1α and found that the protein-coding region of 9 exons encompasses only 4 kb (DNA Cell Biol. 16:1497, 1997). We now describe the genetic analysis of VDDR-1 using a rapid, one-step genomic PCR tactic. A single pair of oligonucleotides was used to amplify the entire 4 kb genomic fragment containing the protein-coding region, excluding only the 5' and 3' untranslated regions. Because an entire parental allele can be amplified as a single fragment, analysis of parental DNA is not required to establish zygosity. The 4 kb PCR fragment was either subcloned or subjected to direct sequencing without cloning. With either approach, a pair of primers for each exon was then used to determine the sequence from each strand. Using this tactic we have examined the genomic DNA from six VDDR-1 families. The patients carried various premature stop codons or frame-shift mutations that gave rise to truncated proteins that cannot bind heme, thus prohibiting P450 catalytic activity. Examination of parental DNA confirmed zygosity as expected. These data demonstrate that VDDR-1 is caused by a variety of mutations in CYP40, which can now be assessed easily and rapidly by a PCR-based tactic that does not require the examination of parental DNA.