Nuclear Factor I (NFI) proteins constitute a family of dimeric DNA-binding proteins with very similar, possibly identical, DNA-binding specificity. They function as cellular transcription factors and as replication factors for adenovirus DNA replication. Diversity in this protein family is generated by multiple genes, differential splicing, and heterodimerization. To determine the chromosomal position of NFI genes in the human genome, we isolated partial cDNA sequences derived from four independent genes: NFIA, NFIB, NFIC, and NFIX. Corresponding clones of genomic DNA served as probes for fluorescence in situ hybridization on human metaphase chromosomes. The NFIA and NFIB genes map to positions 1p31.2-p31.3 and 9p24.1, respectively. The NFIC and the NFIX genes were both localized to position 19p13.3 in the order centromere-NFIX-NFIC-telomere. Comparison of the position of NFI genes and JUN genes revealed a close physical linkage between members of the NFI and JUN gene families in the human genome.