Relaxin is a peptide hormone which is produced in human reproductive tissues including the ovary and prostate gland. Little is known of the molecular events regulating relaxin gene transcription. We have studied this question using gene transfer of relaxin promoter/reporter gene constructs into a relaxin-expressing cell line. A number of human cell lines expressed relaxin as detected by reverse transcription-PCR. In one of these lines, the prostate adenocarcinoma cell line LNCaP.FGC, relaxin mRNA was also detected by Northern blot analysis. The DNA sequences of the proximal 5′-flanking regions (∼900 nucleotides) of the two human relaxin genes, HI and H2, were determined. Deletion constructs containing portions of the 5′-flanking regions of HI and H2 linked to the bacterial chloramphenicol acetyl transferase reporter gene were prepared. The expression of the reporter gene constructs was analysed in the LNCaP.FGC cell line and the results of these transient transfection assays have led to the identification of positive and negative regulatory regions within the 5′-flanking DNA. A difference in activity of the H1 and H2 gene promoters in this prostate cell line was observed, with the H2 promoter being more active. This situation may mimic that occurring in vivo since the relaxin secreted from the prostate gland into seminal fluid is the product of the H2 gene.