The promoter region of the chicken alpha2(I) collagen gene contains a pyrimidine-rich element that is well conserved in different mammalian species. This sequence can also form an unusual DNA structure as shown by its sensitivity to SI nuclease in vitro and it lies in a region that is DNase I-hypersensitive only when this promoter is active. We have recently reported that fibroblast nuclear proteins, including chicken Y-box-binding protein 1, bind to this single-stranded pyrimidine-rich sequence. Here we report the isolation, from a chick embryo fibroblast cDNA expression library, of a partial cDNA clone encoding a previously unknown protein, designated SSDP (sequence-specific single-stranded DNA-binding protein), that binds this single-stranded sequence. This clone contains 1199 bp of chicken sequence and has a single long open reading frame that encodes 284 amino acid residues. The affinity-purified recombinant protein encoded by this cDNA binds sequence-specifically to the single-stranded pyrimidine sequence. This cDNA sequence lacks significant similarity to any known gene in the data banks, but it is highly conserved in expressed sequence tags derived from both mouse and human. The corresponding amino acid sequence is remarkably conserved, having 97% identity with mouse and human expressed sequences. The corresponding mRNA is approx. 1800 nt in length and is expressed in both fibroblasts and chondrocytes. The high affinity of this protein for this conserved pyrimidine-rich region suggests that it might be involved in the transcriptional regulation of the alpha2(I) collagen gene.