Human DNA-binding proteins, dbpA and dbpB (YB-1), are members of a protein family containing a cold-shock domain, and are regarded as transcriptional regulators. Here, we isolated genomic fragments of these genes and characterized their transcriptional regulation. Analysis of lambda phage genomic clones revealed that the dbpA gene consists of 10 exons spanning a 24-kb genomic region. The cold-shock domain, composed of about 70 amino acid residues, is encoded separately by exons 2-5. The exon 6, encoding 69 amino acid residues, was found to be an alternative exon. Northern-blot analysis showed that both genes were highly expressed in skeletal muscle and heart compared with in other tissues. The dbpA gene contains no typical TATA box or CAAT box at the immediate 5' region, but a sequence similar to an initiator consensus sequence was revealed at a major transcription-start site. A transient expression assay using the chloramphenicol acetyltransferase reporter gene revealed that the sequence located at positions -17 to +70 relative to the major transcription-start site was critical for promoter function. Within this region, the consensus sequence for serum-response element, CC(A/T)6GG, is present at positions -13 to -4 in addition to the initiator sequence. Immunofluorescence showed the cellular localization of dbpA to be both in the cytoplasm and nucleus, particularly at the perinuclear region. In situ hybridization demonstrated the localization of the dbpA gene on chromosome 12 band p13.1, whereas dbpB-(YB-1)-related genes were dispersed on many chromosomes with strongest hybridization signals on chromosome 1. All 16 dbpB (YB-1) clones, isolated from the same genomic library used for dbpA genomic cloning, were processed genes because of their intronless structures and multiple mutations. One of these processed genes possesses an open reading frame, which encodes most of the amino acid residues of dbpB (YB-1). These results indicate that dbpA and dbpB (YB-1) genes evolved in different fashions after deviation from a common ancestral gene.