W e have examined the expression and secretion of endogenous parathyroid hormone-like peptide (PLP) in primary cultures of normal human keratinocytes. In response to growth factors and fetal bovine serum, PLP mRNA expression and immunoreactive PLP release into conditioned medium was rapidly increased (within hours) whereas these effects were inhibited by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. These early responses were not influenced by raising the medium calcium concentration from 0.15 to 1.0 mM. In contrast, increasing the medium calcium concentration to 1.0 mM, addition of 1,25(OH)2D3, or a combination of both, resulted in a delayed augmentation (after several days) in PLP production which was associated with an increase in cellular differentiation as assessed by production of high molecular weight keratin. To investigate whether these factors were acting at the level of transcription of the PLP gene, a series of vectors were prepared by fusing segments of the 5' flanking region of the rat PLP gene to a growth hormone reporter gene. Transient transfection of these constructs into cultured keratinocytes and measurement of immunoreactive growth hormone in the medium showed that a region stimulated by growth factors is located in a 1.9-kb fragment of the 5' flanking region and that a PLP gene promoter region less than 1.2 kb and greater than 0.3 kb upstream of the cap site contains cis-acting elements which respond positively to serum, and negatively to 1,25(OH)2D3. These combined studies demonstrate that, in normal human keratinocytes, growth factors may acutely stimulate PLP mRNA levels and PLP release, whereas 1,25(OH)2D3 inhibits these responses. At least part of these effects are at the level of gene transcription. Additionally, PLP synthesis and release are enhanced under conditions in which keratinocyte differentiation is induced.