PTH-related protein (PTHrP) is the principle mediator of the syndrome of humoral hypercalcemia of malignancy and has potential paracrine actions on smooth muscle, epithelial cell growth, and placental calcium transport. The human PTHrP gene is complex: a combination of three promoters, one 5' alternative splicing event and alternative 3' splicing, which produces three PTHrP isoforms (139, 141, or 173 amino acids), results in multiple PTHrP messenger RNA (mRNA) species. We employed the RT-PCR technique to identify promoter usage and splicing patterns in a range of human cell lines. Cell line-specific utilization of the promoters and the 3' alternative splicing pathways was detected among bone, breast, kidney, and lung cell lines, although each cell line could potentially produce the three PTHrP isoforms. We also determined whether some of the known regulators of PTHrP differentially modulate promoter usage or splicing patterns. Dexamethasone decreased the abundance of each of the alternative mRNA species. In contrast, epidermal growth factor and transforming growth factor-β treatment increased the abundance of each PTHrP mRNA species, with particularly marked effects on promoter 1- and promoter 2-initiated transcripts, especially those containing exon VII or VIII. Epidermal growth factor treatment was found to alter PTHrP splicing patterns in a manner consistent with increased transcription from promoters 1 and 2 and stabilization of exon VII- and IX-containing transcripts.