The genes for chemokine-like factor (CKLF) and four chemokine-like factor super family members (CKLFSF1-4) are tightly linked on chromosome 16, with only 325 bp separating CKLF and CKLFSF1. We used Northern blotting and RT-PCR to show that these two genes are expressed independently of one another. We then used a novel computational promoter prediction method based on the interaction among transcription factor binding sites (TFBSs) to identify a putative promoter region for the CKLFSF1 gene. Our method predicted a promoter region in the last intron of the upstream gene, CKLF. We PCR amplified the predicted promoter region and used a luciferase assay to show that the region was able to drive the luciferase gene. DNA decoy experiments indicated that 214 bp fragment neighboring the TATA box markedly inhibited CKLFSF1 gene expression. Sequence analysis of the region revealed a typical TATA box (TATATAA) and multiple potential transcription factor binding sites, providing further evidence for this being a functional promoter for CKLFSF1. This work provides the first evidence of a promoter from one gene located in an intron of another.