Insulin-I and -II were purified from stone flounder (Kareius bicoloratus), and their primary structures were determined. The amino acid sequences of insulin-I and -II from stone flounder were identical with those of barfin flounder (Verasper moseri) except for position 2 of both B-chains. Insulin-II of stone flounder had an extension of the two amino acid residues (Pyr-Ala), at the N-terminus of the B-chain. These structural characteristics of insulins from stone flounder support the idea (Andoh and Nagasawa, Zool. Sci. 15: 901, 1998) that insulin-I and -II of flounders arise from a single preproinsulin in each species by proteolytic cleavage at different sites of the signal peptide region, and suggest that this generation system of two molecular forms of insulin is not specific for barfin flounder. In the course of the purification of insulins, somatostatin-14 and two glucagons (glucagon-I and -II) were also purified from the extract of the Brockmann body. The amino acid sequence of somatostatin-14 of stone flounder was identical with those of mammals. Five amino acids were different between glucagon-I and -II of stone flounder. The amino acid sequences of both glucagons were highly conserved among several acanthopterygian and paracanthopterygian fish. These results suggest that their common ancestral species had both glucagons.