A series of benzo[h][1,6]naphthyridine and azepino[3,2-c]quinoline derivatives were prepared and evaluated to determine the necessary requirements for high affinity on the 5-HT(4) receptors and high selectivity versus other receptors. The compounds were synthesized by substituting the chlorine atom of benzonaphthyridines and azepinoquinolines with various N-alkyl-4-piperidinylmethanolates. They were evaluated in binding assays with [(3)H]GR 113808 as the 5-HT(4) receptor radioligand. The affinity values (K(i) or inhibition percentages) depended upon the substituent on the aromatic ring on one hand and the substituent on the lateral piperidine chain on the other hand. A chlorine atom produced a marked drop in activity while a N-propyl or N-butyl group gave compounds with nanomolar affinities (1 < K(i) < 10 nM). Among the most potent ligands (3a, 4a, 5a), 4a was selected on the basis of its high affinity and selectivity for pharmacological screening and was evaluated in vivo in specific tests. This compound reveals itself as an antagonist/low partial agonist in the COS-7 cells stably expressing the 5-HT(4(a)) receptor. Derivative 4a also showed in vivo potent analgesic activity in the writhing test at very low doses.