Prolactin (PRL) has been implicated in the development of mammary cancer in rodents and humans. Although PRL and its mRNA have been detected in breast tissues and some mammary cell lines, the role of PRL as an autocrine/paracrine growth factor within the breast is not clear. A second, more distal, promoter has recently been identified in the human PRL gene. We have used reverse transcription-polymerase chain reaction (RT-PCR) to determine whether the distal or the proximal promoter directs expression of the PRL gene in normal and neoplastic breast tissues and in mammary cell lines. Total RNA was isolated from 10 normal and 20 neoplastic breast tissue samples and from 8 mammary cell lines; MDA-MB-231, SK-BR-3, T-47D, MCF10, MCF10T2, and 3 MCF7 derivatives. The RNA was reverse transcribed to cDNA using random hexamers as primers. PCR amplification of the cDNAs was performed, using a variety of PRL-specific primer pairs, and the DNA products were subjected to agarose gel electrophoresis and Southern blotting. The resulting data indicate that the PRL gene is expressed in the majority of both normal and neoplastic breast tissue samples, as well as all of the mammary cell lines. PRL-specific PCR products corresponding to transcripts that originated from the distal promoter were observed in a subset of the normal and neoplastic breast tissue samples and mammary cell lines. Together these data indicate that PRL transcripts in human breast tissues and human mammary cell lines originate, at least in part, from the distal PRL promoter. In addition, data are presented which suggest that PRL transcripts in breast tissues and mammary cell lines may undergo alternative splicing.