A specific and sensitive radioimmunoassay (RIA) was developed for the measurement of sodefrin, a female-attracting decapeptide pheromone of the red-bellied newt, Cynops pyrrhogaster. Sodefrin synthesized according to the amino acid sequence of native sodefrin, isolated from the abdominal glands of the cloaca of the male newt, was used as a reference standard. An antiserum to sodefrin was produced by immunizing a rabbit with synthetic sodefrin that was extended on its C-terminus with Cys coupled to hemocyanin. For the radioligand, sodefrin N-terminally extended with Tyr was used. An aqueous extract of the abdominal glands of C. pyrrhogaster produced a displacement curve parallel to the sodefrin standard, whereas that from the sword-tailed newt (Cynops ensicauda) showed no inhibition of binding in this RIA. The sensitivity of the RIA was 30.5 +/- 3.4 pg/100 microliters assay buffer. Intraassay and interassay coefficients of variation were 1.6 and 4.5%, respectively. The RIA was used to determine sodefrin levels in the abdominal gland of the male newt. Hypophysectomy and castration greatly reduced the sodefrin content. Administration of testosterone propionate (TP) to the hypophysectomized and castrated newt increased the pheromone content in the abdominal gland. A combination of prolactin (PRL) and TP elevated the sodefrin content markedly, while PRL alone scarcely affected it. Immunoreactive sodefrin was observed in the epithelial cells of the abdominal gland of both hormone-treated and saline-injected newts. Among them, groups treated with PRL plus TP and TP alone exhibited strong immunoreactivity in their abdominal gland compared with PRL-treated and saline-injected groups.