Double-stranded cDNA was synthesized from partially purified winter flounder antifreeze mRNA and inserted into the endonuclease Pst I site of plasmid pBR322 by the poly(dG) poly(dC) homopolymer extension technique. The recombinant plasmids were used to transform Escherichia coli. Clones containing antifreeze cDNA inserts were identified by the hybridization-selection technique. One of the inserts, 380 nucleotides in length, was digested with endonucleases Sau3AI and Hinfl, which cleaved the insert into three fragments. The nucleotide sequences ofthese fragments were determined. The cDNA contains the entire coding sequence for a possible antifreeze peptide, including the leader sequence. The predicted amino acid sequence is similar to but not identical to one of the known sequences of antifreeze peptide. Within the cDNA are three segments of repeating sequences. The basic repeating sequence of 11 amino acids is maintained in the amino acid sequence coded by the cDNA and in the antifreeze peptide.Winter flounder (Pseudopleuronectus americanus) is found in the shallow waters off the Atlantic coast of North America. In distribution it ranges as far south as Georgia and as far north as Labrador (1). Serum from the northern populations is enriched with a group ofsmall peptides (antifreeze peptides) that depress the freezing point of the body fluid and provide protection during winter months (2, 3). These antifreeze peptides are not found in the serum of southern populations (4). Seasonal variations in the concentration of antifreeze peptides occur, with highest concentrations in the winter months (5, 6). Laboratory acclimation studies have indicated that both temperature and photoperiod control the synthesis and degradation ofantifreeze peptides (7). Peptides responsible for the freezing point depression were isolated and purified from the serum of winter specimens (3,8). Three peptides (nos. 1-3) with freezing point depressing activity have been identified. They are small in size (molecular weights, 3500 to approximately 7000) and simple in amino acid composition. They contain nine different amino acids; 66% of the total amino acids are alanine (8). The amino acid sequence ofthe smallest peptide, no. 3, and that ofthe first 10 amino acids of peptides 1 and 2 have been determined (8). The sequence of peptide 3 can be divided into three segments 11 amino acids in length, each beginning with threonine. The predominant nonpolar residues are alanine. Circular dichroism spectra have indicated that over 80% of the peptides are in the form ofan a-helix (9), and viscosity studies have shown that they are rod-shaped molecules (10). A molecular model with a-helical configuration has been constructed for peptide 3 (8). In this model all polar side chains are coplanar and separated by 4.5 A, the distance between neighboring oxygen atoms in the ice lattice. The structure also aligns all the nonpolar side chains on the opposite side of the molecule. From this model, it is conceivable that antifreeze peptides can bind t...