Sheep stalk median eminence fragments were lyophylized, extracted and filtered through a column of Sephadex G-25. The fractions were then assayed for the presence of LHRH by radioimmunoassay (RIA) and bioassayed for FSH and LH-releasing activity following their IV injection into ovariectomized, estrogen progesterone-blocked rats. The radioimmunoassayable LHRH emerged from the column at the same position from which it emerged many years before when LH was measured by bioassay. This same region also contained the LH-releasing activity as measured by bioassay. FSH-releasing activity was present in two tubes just preceding the emergence of the bio- and immunoassayable LHRH. The activity was highly significant and there was no LH-releasing activity in the fractions. They contained much less LHRH as determined by RIA than is sufficient to evoke LH release in this assay. The FSH-releasing activity was recovered in the same fractions in which it was found many years ago with this same assay but with measurement of plasma FSH by bio-rather than immunoassay as employed here. A dose-related release of LH was obtained by injection of LHRH in this assay but there was no significant FSH release even with a dose of 27 ng of LHRH per rat. To determine if one of the LHRHs of lower forms might be FSH-RF, Chicken I and II LHRH and Salmon LHRH were also assayed for FSH- and LH-releasing activity. Each of these peptide possessed LH-releasing activity, albeit much less than that of the mammalian peptide but had no FSH-releasing activity whatsoever.(ABSTRACT TRUNCATED AT 250 WORDS)