Bullfrog insulin was extracted and purified from the pancreas of Rana catesbeiana adults using gel filtration and reverse phase high performance liquid chromatography. Amino acid analysis of bullfrog insulin revealed 52 amino acids instead of the most common number of 51. Hagfish insulin is the only other known insulin with 52 amino acids. The most unique feature of bullfrog insulin is a two amino acid extension on the amino terminus (A1) of the A chain. This is the only insulin to date that has an extension at this position. In mammals, the deletion of A1 glycine or addition of a bulky group greatly reduces the biological activity of the insulin. Most of the structurally important amio acids have been conserved, thus preserving the integrity of the three-dimensional molecule;Bullfrog and porcine insulin increase protein secretion from bullfrog adult and three developmental stages of tadpole hepatocytes in a totally defined, serum-free culture system. The hormone slightly stimulates protein secretion by premetamorphic and early prometamorphic tadpoles. Late prometamorphic tadpoles respond to bullfrog and porcine insulin with higher concentrations of secreted protein than either of the two previous developmental stages. Insulin treated adult hepatocytes secrete significantly higher (p < 0.05) concentrations of protein than any of the tadpole stages. In addition, adult hepatocytes respond to bullfrog insulin with higher concentrations (p < 0.01) of secreted protein when compared to porcine insulin;[superscript]35S-methionine and [superscript]35S-cysteine were added to the culture medium for twelve hours. Proteins secreted into the medium were separated using SDS polyacrylamide linear gradient gels. Densitometer scans of autoradiograms did not show an increase in any specific proteins, but did show a generalized increase in all secreted proteins for both adults and tadpoles. Albumin increased from 12% of total secreted protein during early prometamorphosis to 40% of total during late prometamorphosis, and 48% in adults.