Isolation and sequencing of a genomic clone encoding the mouse gene for the relaxin-like factor (RLF), which is endogenously expressed to a high level exclusively in Leydig cells, indicated that similar sequences were also present at the 3' end of the mouse JAK3 gene, a gene expressed predominantly in lymphoid tissues. More extensive Southern blot, polymerase chain reaction and sequencing analyses showed that the published mouse sequence for exon 23 of the JAK3 gene in fact comprises two exons, 23A and 23B, separated by an additional novel intron of 2.2 kb, and that within this intron the promoter and exon 1 of the mouse RLF gene are encoded. The two overlapping transcripts appear to use different polyadenylation signals in the common 3' untranslated region of exon 23B. Transient transfection of different RLF promoter reporter constructs into Leydig, Sertoli, granulosa and kidney cell lines indicate that as little as 0.7 kb of the region upstream of exon 1 of the RLF gene, and within the novel intron 22 of the JAK3 gene, is sufficient to account for cell-specific expression of the RLF gene. This promoter region is specifically hypomethylated in Leydig cells compared to non-expressing tissues.