Parathyroid hormone-like protein (PLP) is expressed in a wide variety of cancers and exerts diverse biological effects in addition to hypercalcemia. We studied the expression of the gene for PLP in cancer cell lines derived from different tissues that produce PLP. We used the polymerase chain reaction to evaluate the PLP mRNA species produced in these various cell lines by differential transcription initiation and alternative splicing pathways. A series of exon-specific oligonucleotide primers that hybridize to DNA sequences adjacent to exon-intron splice junctions throughout the PLP gene were designed. These primers were used to evaluate steady state levels of PLP mRNA species. Our analysis with promoter-specific primers demonstrated expression from all three putative transcription start sites of PLP, designated P1, P2, and P3. P2-initiated transcripts were present in all of the cell lines, whereas the presence of P1- and P3-initiated messages were cell line specific. Our analysis with carboxy-terminal coding-specific primers demonstrated the utilization of the alternative splicing pathways that produce all three mature PLP polypeptides, PLP-139, -1-173, and -1-141. The 1-139 mRNA species was found in all of the cell lines, whereas the 1-141 and 1-173 mRNA species were cell line specific. These studies demonstrate the prevalence of the P2-initiated mRNA and the PLP1-139 alternative splicing pathway in the tumor cell lines studied and suggest that other pathways of PLP gene expression may be regulated in a cell line-dependent manner. The particular form of PLP expressed by a given cell can influence its biological effects.