A genomic DNA clone containing the 5'-end of the rat topo II alpha gene was isolated and the intron/exon structure of a 4.0 kb region encompassing the translation initiation site was determined. Multiple transcription initiation sites were found at positions -128, -110, and -100 bp upstream of the ATG codon. A minimal promoter region extending from -192 to the translation initiation codon was identified on transient expression analysis. This region lacks a TATA motif, is moderately GC-rich and contains a high number of CpG dinucleotides, which is characteristic of a housekeeping gene promoter. The fragment extending to position -242 exhibited maximal promoter activity. Putative regulatory elements were delineated within and immediately upstream of the minimal promoter region. On gel retardation and DNase I footprint analyses, two regions, between positions -195 to -159 which interact with protein factor(s) were identified. The minimal promoter region of the rat topo II alpha gene showed high sequence homology with that of human topo II alpha. In a 250 bp region upstream of the translation initiation site, the sequence identity was about 70%. The basic structure of the regulatory region of the rat topo II alpha gene was found to be similar to that of the human counterpart.