The primary structure of the T4 single-stranded DNA-binding protein coded by gene 32 has been determined by manual and autoated sequencing of peptides derived from partial proteolysis, cyanogen bromide cleavage, and digestion with trypsin, chymotrypsin, and staphylococcal protease. Tryptic digestion of citraconylated or succinylated gene 32 protein yields five peptides containing 4, 27, 42, 65, and 163 residues, which can be separated by Sephadex chromatography. Each of the tryptic peptides was subjected to automated sequencing and, if necessary, more extensive cleavage. The intact protein contains 301 amino acids, has a molecular weight of 33,487, and can be specifically cleaved at lysines 21 and 253 by limited trypsin digestion. Previous studies have shown that the "B" region (residues 1-21), which has a charge of +4, is important for the protein-protein interactions involved in gene 32 protein self-association and cooperaive binding to single-stranded DNA. The "A" region (residues 254-301) has been implicated in controlling the helix-destabilizing "activity" of gene 32 protein and in interacting with other T4 DNA replication proteins. The A region has a charge of -10 and, in addition, contains two unusual stretches of four serine residues separated by glycine 284. The region between positions 73 and 115 contains 75% of the tyrosine residues and may be important for DNA binding.