Cultured primary human keratinocytes were the first non-cancer-derived cell type reported to produce the humoral hypercalcemia factor, parathyroid hormone-related protein (PTHrP). Emerging evidence suggests that only a subset of keratinocytes produce high levels of PTHrP in vivo. We found that the PTHrP mRNA content of intact human skin was minimal, whereas transcripts were easily detectable in primary keratinocytes derived from those skin samples. We hypothesized that conditions associated with growth in culture activated PTHrP gene expression in primary keratinocytes. In culture, keratinocytes produce a number of epidermal growth factor (EGF)-like ligands (transforming growth factor-alpha, heparin binding-EGF and amphiregulin) and their receptor, ErbB1. Treatment of keratinocytes with a specific erbB1 inhibitor (PD153035) reduced PTHrP mRNA levels by >80% in rapidly growing keratinocytes. Treatment of keratinocytes with reagents that neutralize amphiregulin reduced PTHrP mRNA levels by approximately 60%. Blockade of erbB1 signaling reduces transcription from the endogenous PTHrP P3-TATA promoter. The Ets transcription factor-binding site, 40 bases upstream of the P3 promoter, is required for baseline expression of PTHrP reporter gene constructs in keratinocytes; in addition, cotransfection of Ets-1 and Ets-2 expression vectors activate the reporter gene constructs. Finally, disruption of both ras and raf signaling reduce reporter gene expression by 80%, suggesting that ErbB1 signaling is mediated by the classic ras/MAP kinase pathway. These findings suggest that acquisition of EGF-like ligand expression has the potential to substantially activate PTHrP gene expression in the epidermis.