The genes for CKLFSF1 (chemokine-like factor super family member 1) and CKLFSF2 (chemokine-like factor super family member 2) are very closely linked, within 312 bp of each other. Here, we present evidence that the last intron/exon region of the CKLFSF1 gene contains a novel eukaryotic promoter capable of directing the expression of the downstream gene, CKLFSF2. We identified two segments of the upstream region of the CKLFSF2 gene, 2146 bp (-2134/+12, relative to ATG +1) and 1483 bp (-2134/-652), that were capable of efficiently driving expression of a linked reporter gene upon transient transfection into several kinds of cell lines. The 1483 bp segment exhibited more than a two-fold increase in luciferase activity relative to the 2146 bp segment. By analyzing 5'-deletion mutants of the 1483 bp segment, we identified a 195 bp segment (-846/-625) located in the last intron/exon region of the CKLFSF1 gene that was critical for promoter activity. DNA decoy experiments revealed that a 122 bp (-846/-725) fragment markedly inhibited CKLFSF2 mRNA transcription. Furthermore, we found that the putative promoter region of the CKLFSF2 gene is separated from the transcription start site by about 500 bp. Accumulating reports suggest that introns have many functions, including the modulation of regulation and structure. This work provides evidence that a eukaryotic gene promoter sequence from one gene located in an intron/exon of another.