The pancreatic beta-cell is the only cell in animals that expresses the insulin gene and secretes insulin protein. We have found copious release of immunoreactive and bioactive insulin into the medium from the primary culture of carp adipocytes. Glucose augmented this release to more than 2-fold, and glucose transporter, Glut2, was detected in these cells. These all reflect characteristics of a pancreatic beta-cell. The expression of the adipocyte-specific flotillin gene, the presence of peroxisomal proliferator-activated receptor gamma and Glut4, and the colocalization of insulin and leptin confirmed the identity of these cells as adipocytes. Purified carp adipocyte insulin (AdpInsl) comigrated with porcine and bovine insulin in SDS-PAGE, indicating the similarity of their molecular sizes (5.5 kDa). AdpInsl strongly reduced hyperglycemia in streptozotocin-induced diabetic rats. It also stimulated significantly higher glucose uptake in carp and hamster adipocytes than porcine insulin. Adipocyte RNA hybridized with rat and zebrafish insulin cDNA showing the expression of the insulin gene in this cell. Using oligonucleotide primers designed on the basis of conserved insulin domain, AdpInsl cDNA was reverse transcribed, cloned, and sequenced. The deduced amino acid sequence of AdpInsl A and B chain exhibited 98% homology with zebrafish and more than 70% homology with human, porcine, and murine insulin. To understand the structure-function relationship between AdpInsl and mammalian beta-cell insulin, we have analyzed the amino acid sequences and three-dimensional structure of AdpInsl. In the critical determinant segment for receptor binding, AdpInsl has His at the A8 position instead of Thr in human and porcine insulin, and this attributed greater biological activity to AdpInsl. Our results show that carp adipocyte is a unique cell. As an insulin target cell it can express the insulin gene and secrete highly active insulin protein; thus, it may serve as a natural alternative to pancreatic beta-cell insulin.