Type-I collagen, the predominant component of extracellular matrix, is a triple-helical protein consisting of two α1 polypeptides and one α2polypeptide. Expression of α1 and α2 procollagen genes is co-ordinately regulated under both normal and various pathological conditions. However, the basis of this co-ordinate regulation is not well known. YB-1b, a Y-box protein, has been shown to bind to the polypyrimidine tract present in the α2 procollagen gene. Here, we show that chk-YB-1b, a YB-1 homologue, binds in a single-strand-sequence-specific manner to the highly conserved pyrimidine-rich sequences in both α1(I) and α2(I) procollagen promoters from different species, as demonstrated by electrophoretic-mobility-shift assays and by DNaseI footprinting experiments. Transiently transfected and retrovirally expressed antisense oligonucleotides directed against chk-YB-1b specifically inhibited the α1(I)procollagen promoter-driven transcription in cultured fibroblasts. Considering these data and the fact that the chk-YB-1b binding site is one of the few sites between α1(I)and α2(I)procollagen promoters that is conserved from chicken to human, it is proposed that chk-YB-1b may be involved in co-ordinate expression of these two collagen genes.