The complete nucleotide sequences of cDNAs encoding bovine and feline preprogastrins have been cloned from the antral mucosa mRNA. The gastrin mRNA of each animal encodes a preprogastrin of 104 amino acids consisting of a signal peptide, a prosegment of 37 amino acids, and a gastrin 34 sequence, followed by a glycine (the amide donor). The cleavage following a pair of lysine residues yields gastrin 17. We found that pairs of arginine residues flanking gastrin 34, the typical processing site sequence of all other preprogastrins and many peptide hormones, were arginines in the bovine preprogastrin, but the first basic amino acid pair had changed to Arg-Trp (57-58 residues) instead of Arg-Arg in the feline preprogastrin. Comparison of these amino acid and nucleotide sequences with published mammalian sequences showed extensive homology in the coding (63 to 73% amino acid identity) and in the untranslated regions (67 to 89% identity). Prosequence, the most variable region, shows greater amino acid difference between bovine and human preprogastrin (54% identity), and between bovine and rat preprogastrin (54% identity) than between other species (62 to 82% identity).