Conformationally restricted analogues of atriopeptin(103-125)amide were prepared by synthesizing novel bicyclic peptides in which a second disulfide bridge linking residues 108 and 117 was introduced. These syntheses were shown to proceed with no significant scrambling of the disulfide bonds and demonstrated that structurally defined bicyclic analogues of atrial peptides could be easily prepared. The conformationally restrained analogues described here were found to be biologically active with potencies (EC50s) ranging from 0.05 to 3 microM. In addition, these bicyclic peptides (and many of the monocyclic precursors) were found to bind selectively to a class of specific tissue binding sites that have not been shown to be associated with any known second messenger system (NVR binding sites). Since affinity for the receptor class linked to vasorelaxation was negatively affected by the conformational restrictions described here, binding of atrial peptides to this class of receptors appears to have more specific conformational requirements than does binding to the NVR sites.