Previous evidence has suggested that the human PTH-related peptide (PTHRP) gene uses two promoters, one a short down-stream element lying immediately between two 5' exons (1 and 2) and a second lying in an unknown up-stream location. We approached identification of the up-stream element in three steps. First, Northern analysis carried out using progressively 5' fragments of the gene as probes identified a candidate region some 2.5 kilobases up-stream of exon 1. Second, a battery of overlapping 5' cRNA probes was used in RNase protection experiments to identify two previously unrecognized exons, 212 and 93 basepairs in length (termed exons 1A and 1B to distinguish them from the previously designated exon 1, which was termed exon 1C). Third, primer extension experiments were performed with oligonucleotides complementary to each of the 5' exonic sequences. These experiments identified a transcription start site up-stream of exon 1A and also demonstrated that the 5' exons of the PTHRP gene could be spliced together in several combinations. The up-stream promoter element contains a TATA box, but does not otherwise resemble the down-stream PTHRP gene promoter or the PTH gene promoter. We conclude that the human PTHRP gene contains eight exons spanning more than 15 kilobases of genomic DNA, with promoter elements lying immediately up-stream of exons 1A and 2. The identification of these elements will permit functional analysis of their roles in mediating tissue- and tumor-specific PTHRP gene expression.