The expression of non-pituitary human PRL is initiated at a unique 5' untranslated exon located approximately 5.7 kb upstream of the pituitary-specific transcriptional start site. Unlike pituitary PRL expression, transcriptional regulation from the upstream promoter does not rely on the POU-homeodomain protein Pit-1. We have used DNase I mapping of chromatin from PRL-producing and non-producing human lymphoblastoid cell lines to identify hypersensitive sites unique to the PRL expressing phenotype. Analysis of 22 kb of 5' flanking DNA revealed DNase I hypersensitive sites in intron A-1 separating the pituitary from non-pituitary specific transcription start site which were only detected in the PRL-producing cell line. Transient transfection showed strong transcriptional activity directed by this region only in the antisense orientation and in a non cell-type specific manner. Transfection experiments with deletion mutants of 5259 bp of the non-pituitary PRL promoter region also revealed promoter activity not restricted to the PRL expressing phenotype. These data suggest that non-pituitary PRL gene expression may be regulated by elements located in intron A-1 and that recapitulation of cell-specific expression requires a unique cellular context and chromatin assembly.