Thein vivo andin vitro potency of native and modified forms of gonadotropin releasing hormone (GnRH) to release gonadotropin (GtH) was studied inSparus aurata and correlated with their relative susceptibility to degradation by cytosolic-bound enzymes of the pituitary, kidney, and liver. Salmon (s) GnRH and luteinizing hormone-releasing hormone (LHRH) are equipotent whereas analogs of these peptides ((D-Arg(6)-Pro(9)NET)-sGnRH, (D-Ala(6)-Pro(9)NET)-LHRH, (D-Trp(6))-LHRH) are superactive in inducingin vivo GtH release (at 10 µg/kg body weight). In anin vitro superfusion system of pituitary fragments all analogs are equipotent to the native peptides (at 10(-10) to 2.5 × 10(-7)M). sGnRH and LHRH are rapidly degraded by cytosolic peptidases of the pituitary, liver, and kidney. The preferred site of cleavage is the Tyr(5)-Gly(6) bond. Substitution of the position 6 glycine by D-amino acids renders the 5-6 bond resistant to degradation and shifts the main site of cleavage to the Pro(9)-Gly(10)NH2 bond. Substitution at position 6 (as above) and at position 10 with Pro(9)NET results in analogs that are resistant to degradation. We propose that enzymatic cleavage terminates GnRH bioactivityin vivo and thus increased resistance to degradation is a major determinant of GnRH analog superactivity.