The Australian lungfish Neoceratodus forsteri, a facultative air breather, is considered to be the most primitive of the extant Dipnoi and so occupies a uniquely important evolutionary position in the transition from fish to tetrapods. Insulin was isolated from an extract of the pancreas of N. forsteri and its primary structure established as: A-Chain, Gly-Ile-Val-Glu- Gln-Cys-Cys-His-Thr-Pro10-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys20-Asn- Glu-Thr-Glu; B-Chain, Ala-Ala-Val-Asn-Gln-His-Leu-Cys-Gly-Ser10-His-Leu- Val-Glu-Ala-Leu-Tyr-Phe-Val-Cys20-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Leu-Pro- Lys30-Gly. This amino acid sequence is more similar to that of human insulin than to insulins from present-day amphibians. All the residues in human insulin that are considered to be important in receptor binding, dimerization, and hexamerization are conserved in lungfish insulin except for the substitution (Leu → Phe) at the position corresponding to B17 in human insulin. Consistent with the assertion that the Dipnoi is a monophyletic group, insulins from N. forsteri and from the African lungfish Protopterus annectens contain extensions to the C-terminus of the A-chain and to the N- terminus of the B-chain that have not been found in other sarcopterygian species. However, the unusual amino acid substitutions found in insulin from P annectens (e.g., GlyB21 → Ala, GluB22 → Asp, and ArgB23 → ASn) are not present in N. forsteri insulin, suggesting that they occurred in the Protopterus lineage after divergence of the genera.