The synthesis and the biological characterization of novel highly selective pyrroloquinoxaline 5-HT(3) receptor (5-HT(3)R) ligands are described. In functional and in vivo biological studies the novel quinoxalines modulated cardiac parameters by direct interaction with myocardial 5-HT(3)Rs. The potent 5-HT(3)R ligands 4h and 4n modulate chronotropy (right atrium) but not inotropy (left atrium) at the cardiac level, being antagonist and partial agonist, respectively. Preliminary pharmacokinetic studies indicate that (S)-4n and 4a, representatives of the novel 5-HT(3)R ligands, possess poor blood-brain barrier permeability, being the prototypes of peripherally acting 5-HT(3)R modulators endowed with a clear-cut pharmacological activity at the cardiac level. The unique properties of 4h and 4n, compared to their previously described centrally active N-methyl analogue 5a, are mainly due to the hydrophilic groups at the distal piperazine nitrogen. These analogues represent novel pharmacological tools for investigating the role of peripheral 5-HT(3)R in the modulation of cardiac parameters.