Differential screening of a cDNA library prepared from chicken mitochondrial DNA-less cells led to the isolation of the acidic phosphoprotein PO, as inferred from similar sequences from human, rat and mouse deposited in databases. The chicken PO protein is highly conserved among vertebrates. Nucleotide identities between the chicken and mammalian cDNA sequences are greater than 94%. Amino acid sequence identities greater than 92% are observed between the chicken protein and its mammalian counterparts, and when changes to conservative amino acids are considered, similarities range from 99.4 to 100%. The gene evolved mainly by silent transitions occurring at the third codon position. The highly antigenic carboxy-terminal sequences of mammalian PO proteins are identical to that of the chicken. Southern blot analysis of restricted DNA from chicken embryo fibroblasts suggests that only one gene encoding PO exists in the chicken genome. By Northern analysis, the PO probe detects a major RNA species 1.1-kilobases long, and a minor species 4.0-kilobases long which has no equivalent thus far in mammals.