In order to explore the structural requirements for high 5-HT1A affinity, a series of aryl-substituted N1-phenylpiperazines were synthesized and evaluated for their ability to displace [3H]-8-OH-DPAT from its specific binding sites in rat frontal cortex homogenates. We found 2-methoxy substitution to be favorable, while 4-methoxy substitution was detrimental for 5-HT1A affinity. Substitution with annelated rings at the 2,3-positions was highly favorable for all investigated compounds, with the exception of a pyrrole ring. All other substitutions, except fluoro, in this class of heterobicyclic phenylpiperazines decreased affinity in the order: ortho > para > meta. The loss of affinity in the ortho and para positions is probably due to steric factors: the substituents either cause steric hindrance with the receptor or prevent the compound from adopting the appropriate conformation for binding to the 5-HT1A receptor. Conformational analysis combined with structure-affinity relationships (SAR) indicates that our arylpiperazines may bind at the 5-HT1A receptor in a nearly coplanar conformation. Observed interactions of the compounds in our 5-HT1A receptor model appeared to be in agreement with SAR data. The aromatic part of the arylpiperazine moiety has pi-pi interactions with the aromatic residues Trp161 and Phe362 in helices IV and VI, respectively. The positively charged protonated basic nitrogen forms a hydrogen bond with the negatively charged Asp116 in helix III. The ammonium-aspartate complex is surrounded by aromatic residues Trp358 and Phe361 in helix VI. A lipophilic pocket is formed by Phe362, Leu366 (both helix VI), and the methyl group of Thr200 (helix V). In agreement with the model, addition of a methyl substituent to the structure of the benzodioxine analogue 12 in this region, yielding 13, is favorable for 5-HT1A receptor affinity. Unfavorable positions for substitution with bulky groups, like the 3- and 4-positions in the benzofuran compound 14, are explained by steric hindrance with the backbone atoms of helix V. Thus, we were able to rationalize the 5-HT1A SAR of existing N1-phenylpiperazines, as well as a series of newly synthesized bicyclic heteroarylpiperazines, in terms of receptor-ligand interactions. Several of these N4-unsubstituted compounds had affinities in the low-nanomolar range.