Hyperactive analogues of luteinizing hormone-releasing hormone (LH-RH) are believed to derive their properties from either increased binding affinity to anterior pituitary receptor sites or through decreased susceptibility to enzymic degradation. To investigate the latter suggestion and to examine the possible sites of action of hypothalamic peptidases inactivating LH-RH, D-Ser(TBU)6-EA10-LH-RH and D-Leu6-EA10-LH-RH, which are known to have considerably greater activity than LH-RH, were incubated with a hypothalamic supernatant fraction containing active peptidases degrading LH-RH, and their gonadotrophin-releasing ability after incubation with the enzymes was tested in normal, adult male rats; LH-RH was also tested in the same way. From a comparison of the relative losses of biological activity, both the LH-RH analogues treated proved to be more resistant to the hypothalamic peptidases than LH-RH itself: the D-Leu6-EA10-LH-RH retained its gonadotrophin-releasing activity longer than the D-Ser(TBU)6-EA10-LH-RH. These findings indicate that increased activity of the analogues may, in part, be due to increased resistance to enzymic inactivation and suggest initial sites of cleavage at the Gly-Leu and Pro-Gly NH2 bonds in the LH-RH decapeptide by the hypothalamic enzymes. Studies on the action of peptidases on LH-RH and its analogues may yield useful information in the design of peptides with increased biological activity.