Sea turtle prolactin (PRL) was isolated in a highly purified state from sea turtle pituitary side fractions obtained from other studies and some of its biological, chemical, and immunological properties were determined. Sea turtle PRL is a protein of 22-24 kDa [sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis]. Its sole amino terminal amino acid residue is leucine. The amino acid composition of sea turtle PRL is similar to ovine PRL and is characterized by a high content of aspartic acid (20 residues), glutamic acid (34 residues), serine (19 residues), and leucine (24 residues). It possess three disulfide bonds and 2 tryptophan residues which is also characteristic of many species of PRL. As with PRLs of other species, it displayed multiple stained bands in disc gel electrophoresis, at pH 8.3. Biologically, sea turtle PRL was active in pigeon crop-sac assay but the dose-response characteristics were nonparallel when compared to ovine PRL. An antiserum against sea turtle PRL was raised in rabbit and a homologous radioimmunoassay was developed with a sensitivity of 2.8 ng for sea turtle PRL. Sea turtle gonadotropins did not cross-react, but sea turtle growth hormone showed a 5% cross-reactivity. Pituitary extracts from other species of turtles displayed parallel inhibition curves to the sea turtle PRL. Extracts and prolactin preparations from several birds, snakes, alligator, and marsupials cross-reacted, but in a nonparallel fashion. Bullfrog pituitary extract and Tilapia PRL showed no cross-reaction at high doses. Several purified mammalian PRLs (pig, sheep, human, horse, dog) showed minimal or no ability to cross-react in the RIA.