Prolactin (PRL) is a member of the growth hormone gene family that is specifically expressed in the lactotroph cells of the anterior pituitary. Whole cell extracts have been prepared from cultured GH3 rat pituitary tumor cells to study lactotroph-specific expression of the rat PRL (rPRL) gene in an in vitro transcription assay. The human alpha 1-globin and Rous sarcoma virus promoters efficiently initiate transcription in both HeLa and GH3 cell extracts, whereas the rPRL promoter containing 425 base pairs of 5' flanking DNA is active only in GH3 pituitary cell-free extracts. Transcription of the rPRL gene was reconstituted in HeLa cell extracts by the addition of GH3 cell extracts. DNase I digestion of the rPRL promoter reveals two protected regions centered at positions -55 (I) and -160 (III) that are GH3 cell-specific and rPRL promoter-selective. These "footprints" overlie a highly conserved 8-base-pair motif, CCTGATAATA. By contrast, footprint II at position -125 is common to both HeLa and GH3 cell extracts and overlies a 15-base-pair sequence found in all members of the growth hormone gene family. Thus, GH3 pituitary cell-free extracts selectively transcribe the rPRL gene and contain cell-specific factors that directly interact with the rPRL promoter. These studies provide useful assays to further identify, purify, and characterize pituitary-specific transcription factors and to address the biochemical mechanisms involved in rPRL gene expression.