Structurally new analogs of the peptidic GnRH receptor antagonist Cetrorelix as well as conformationally constrained cyclized deca- or pentapeptides were synthesized and selected peptides evaluated comprehensively. To understand how structural variations of the antagonistic peptide effect pharmacodynamic properties, binding affinities and antagonistic potencies toward the human and rat GnRH receptor were determined. Whereas large substituents in position 6 of linear peptides are compatible with high binding affinity (K-D < 0.5 nM), all cyclized peptides except the cyclo[3-10] analog D-52391 depicted low binding affinity (K-D > 10 nM). Binding affinity and antagonistic potency in vitro correlated for all peptides and surprisingly no discrimination between human and rat receptor proteins was observed. Since receptor residues W-101 and N-102 are involved in agonist and antagonist binding, equally potent but structurally different antagonists were tested. for binding to the respective W(101)A and N(102)A mutants. In contrast to linear decapeptides, residues N-102 and W-101 are not involved in binding of D-23938 and W-101 is the critical residue for D-52391 binding. We conclude that although equally potent, peptidic GnRH receptor antagonists do have distinct interactions within the ligand binding pocket. Finally, selected antagonists were tested for testosterone suppression in male rats. The duration of testosterone suppression below castration levels differed largely from 1 day for Ganirelix to 27 days for D-23487. Systemic availability became evident as the most important parameter for in vivo efficacy.