We have purified and characterized two molecular forms of insulin from the Brockmann bodies of barfin flounder, Verasper moseri: a normal type of insulin (insulin-I), which consisted of 21 amino acid residues for the A-chain and 30 residues for the B-chain, and a novel type of insulin (insulin-II), which had an extension of two amino acid residues at the N-terminus of the B-chain. The additional two residues at the N-terminus of B-chain of insulin-II were Pyr-Ala which had not yet been reported in vertebrate insulins. Except for these two residues, the amino acid sequence of insulin-II was completely identical with that of insulin-I. Each Brockmann body extract from five individuals contained both insulins, indicating that insulin-I and -II were the products of non-allelic expression. By polymerase chain reaction, only one nucleotide sequence of preproinsulin gene encoding insulin-I and -II was obtained, and the amino acid sequence of A-and B-chains deduced from the nucleotide sequence was identical with that of insulin-I and also insulin-II established by Edman degradation. Furthermore, genomic Southern blot analysis using a part of nucleotide sequence of the preproinsulin gene as a probe showed a single positive band in all cases of genomic DNA digested with each of three restriction endonucleases. These results indicate that insulin-I and -II of barfin flounder arise from a single preproinsulin by proteolytic cleavage at different sites of the signal peptide region.