Parathyroid hormone-related protein (PTHRP) is expressed in a large number of tumors and is the mediator of parathyroid hormone-like effects seen in humoral hypercalcemia of malignancy. The gene coding for PTHRP has been localised to the short arm of chromosome 12. This is at the same region as the oncogene KRAS2, and amplification of KRAS2 has previously been found in human lung cancer. The BEN cell line which is known to express PTHRP was established from a patient who had squamous cell carcinoma of the lung with hypercalcemia. Cytogenetic analysis of the BEN cell line revealed a very complex karyotype with many marker chromosomes. Chromosomal in situ hybridization with biotinylated DNA probes visualized by a biotin-streptavidin-polyalkaline-phosphatase complex was used to analyse two dicentric marker chromosomes containing homogeneously staining regions (hsr) in BEN. The hsr were found to contain amplified PTHRP and KRAS2 at levels of 30-fold and 14-fold per cell, respectively. The higher level of amplification of the PTHRP gene would suggest that PTHRP is the target gene of amplification in the amplicon. This is the first report of gene amplification of PTHRP and in addition its co-amplification with KRAS2.