A complete amino acid sequence has been determined for the UP1 single-stranded DNA binding protein from calf thymus that was first described by G. Herrick and B. M. Alberts [(1976) J. Biol. Chem. 251, 2124-2132]. Peptides required to establish the UP1 sequence were isolated by reversed-phase HPLC of digests produced by endoproteinase Lys-C, trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and cyanogen bromide cleavage of UP1. The purified peptides were coupled to aminopolystyrene prior to solid-phase sequencing. UP1 contains 195 amino acids and has a molecular weight of 22,162. UP1 has a blocked NH2 terminus and contains a single NG,NG-dimethylarginine residue near its COOH terminus. Gas-phase sequencing of tryptic peptides derived from an analogous protein from mouse myeloma cells [Planck, S. R. & Wilson, S. H. (1980) J. Biol. Chem. 255, 11547-11556] revealed that this mouse helix-destabilizing protein shares a high degree of sequence homology with UP1. Of the 59 amino acids in the mouse protein that have so far been found to be homologous with UP1, 48 correspond exactly to sequences found in UP1. Most of the 11 differences that have been found between the sequences of these two proteins are conservative in nature, involving primarily the interchange of chemically similar amino acids. One 9-residue mouse sequence that is not obviously homologous to UP1 may be a result of the larger size of the mouse myeloma protein as compared to UP1. Since none of the UP1 or mouse myeloma helix-destabilizing protein sequence appears to be homologous to that of any previously sequenced protein, we presume that these two proteins represent a distinct class of single-stranded nucleic acid binding proteins that probably play a role in metabolism of single-stranded RNA or DNA in vivo.