In the first report on the chemical structure of a nonmammalian LH-RH, chicken hypothalamic LH-RH was demonstrated to be [Gln8]LH-RH [2-4]. However, these studies and subsequent reports [7,8] did not totally exclude the possibility of a reverse sequence of the two amino acids Leu-Gln. In view of the recently described structure of salmon brain LH-RH as [Trp7,Leu8]LH-RH [9], we undertook to confirm our earlier conclusion that chicken LH-RH is [Gln8]LH-RH and not [Gln7, Leu8]LH-RH. The immunologic, chromatographic and biological properties of natural chicken hypothalamic LH-RH were compared with those of the two synthetic peptides, [Gln8]LH-RH and [Gln7,Leu8]LH-RH. A radioimmunoassay highly specific for [Gln8]LH-RH was developed. Natural chicken LH-RH cross-reacted fully with the antiserum which requires the COOH-terminal Gln8 to Gly10-NH2 for binding, while [Gln7,Leu8]LH-RH showed less than 0.1% cross-reaction. On a high resolution reverse phase high performance liquid chromatography system, natural chicken LH-RH co-eluted with [Gln8]LH-RH and was well separated from [Gln7,Leu8]LH-RH. In a chicken anterior pituitary cell bioassay, natural chicken LH-RH and [Gln8]LH-RH were equipotent in stimulating luteinizing hormone release, while the relative potency of [Gln7,Leu8]LH-RH was 4.4%. These data, in particular the use of a specific [Gln8]LH-RH antiserum, provide conclusive evidence that chicken LH-RH is [Gln8]LH-RH.