Most advanced teleosts lack L-gulono-gamma-lactone oxidase (GULO), a key enzyme required for the biosynthesis of ascorbic acid. However, extant representatives of primitive species including sturgeon and many cartilaginous fishes, are exceptional in their ability to synthesize ascorbic acid de novo. In the present study, full-length GULO cDNAs were isolated from white sturgeon (Acipenser transmontanus) and two shark species belonging to the Triakidae (Triakis scyllium and Mustelus manazo). The open reading frames from all three species contained 440 amino acids and the deduced polypeptides had similar hydropathy profiles, predicted molecular masses and theoretical pI values. These GULO sequences exhibited high amino acid identity (67-97%) with each other, and also shared 61-71% identity with mammalian GULOs. Based on the GULO sequences obtained from these species, we developed degenerate primers for the isolation of partial GULO sequences by RT-PCR from other primitive species including another shark (Mustelus griseus, Triakidae), a spiny dogfish (Squalus acanthias, Squalidae), two ray species (Raja kenojei, Rajidae and Dasyatis akajei, Dasyatidae) and four sturgeons (Acipenser baeri, A. gueldenstaedtii, A. naccarii and A. ruthenus, Acipenseridae). Overall, sequence identities of these amplified GULO segments among primitive species were 63-99% at the nucleotide level and 67-100% at the amino acid level. Considerable numbers of amino acid residues were unique to either fish or mammals, and Acipenseriform species occupied an intermediate position, sharing several residues with either fish or mammalian GULOs. Phylogenetic analyses based on parsimony, distance and likelihood methods of both nucleotide and amino acid sequences resulted in trees that were in agreement with known taxonomy. The transcription and enzyme activity of GULO were kidney-specific when measured by biochemical assay and reverse transcription-PCR.