We have characterized a human genomic clone that contains the 5' coding and 5' flanking sequences of the human parathyroid hormone-related protein gene (PTHrP). The 5' end of the gene contains three exons separated by two small introns of 60 and 165 bp, respectively. The coding region of the PTHrP gene exhibits significant structural homology to the human parathyroid hormone gene (PTH), including the position of at least two introns. However, there is no significant nucleotide sequence homology to the PTH gene within the intragenic region nor in the flanking genomic sequences. The PTHrP gene has been localized, by chromosomal in situ hybridization to bands p11 or p12, on human chromosome 12. Analysis of the 5'-noncoding DNA reveals a complex, putative regulatory region, with multiple potential transcription start points. Nucleotide sequence analysis shows the position of one consensus TATA sequence, at -514 bp, from the start of translation whereas the other regulatory domain is located at least 1 kb further 5' to this consensus TATA sequence. Evidence from the structure of a number of cDNA clones, as well as S1 nuclease and primer extension studies supports the hypothesis that the PTHrP gene contains at least two mRNA transcription start points that define two putative regulatory domains. The result of expression from these different promoters combined with an alternative splicing event would be to produce multiple forms of PTHrP mRNA that differ in the 5'-untranslated region. This analysis of the human PTHrP gene is the first report of a PTHrP gene for any species.