We used structure-function analysis of the core promoter region to elucidate the transcriptional features of the mouse alpha 1(V) collagen gene (Col5a1). The core promoter, which lacks a typical TATA motif and has a high GC content, was defined within the 231 bp immediately upstream from the major transcription start site by transient transfection experiments. In this region, we identified three nuclear-factor binding sites by electrophoretic mobility shift assay: BS1 (-195 to -167), BS2 (-134 to -106), and BS3 (-110 to -80). Oligonucleotide competition and supershift assays revealed that Sp1, CBF, and Sp1-related protein specifically bind to BS1, BS2, and BS3, respectively. The CCAAT-like motif, CAAAT, and flanking sequences are conserved between the mouse and human gene. CBF, which recognizes this motif, activated the Col5a1 promoter, as previously reported for Col1a1 and Col1a2. Furthermore, overexpression of a wild-type and mutant forms of CBF-B subunit altered this activity. These results suggest that CBF is a key factor in the coordinated expression of type I and V collagen genes.