BACKGROUND AND PURPOSE: Human and rat 5-HT(7) receptors were studied with a particular emphasis on the molecular interactions involved in ligand binding, searching for an explanation to the interspecies selectivity observed for a set of compounds. We performed affinity studies, molecular modelling and site-directed mutagenesis, with special focus on residue Phe(7.38) of the human 5-HT(7) receptor [Cys(7.38) in rat]. EXPERIMENTAL APPROACH: Competition binding studies were performed for seven 5-HT(7) receptor ligands at three different 5-HT(7) receptors. The functional behaviour was evaluated by measuring 5-carboxytryptamine-stimulated cAMP production. Computational simulations were carried out to explore the structural bases in ligand binding observed for these compounds. KEY RESULTS: Competition experiments showed a remarkable selectivity for the human receptor when compared with the rat receptor. These results indicate that mutating Cys to Phe at position 7.38 profoundly affects the binding affinities at the 5-HT(7) receptor. Computational simulations provide a structural interpretation for this key finding. Pharmacological characterization of compounds mr25020, mr25040 and mr25053 revealed a competitive antagonistic behaviour. Compounds mr22423, mr22433, mr23284 and mr25052 behaved as partial agonists. CONCLUSIONS AND IMPLICATIONS: We propose that the interspecies difference in binding affinities observed for the compounds at human and rat 5-HT(7) receptors is due to the nature of the residue at position 7.38. Our molecular modelling simulations suggest that Phe(7.38) in the human receptor is integrated in the hydrophobic pocket in the central part of the binding site [Phe(6.51)-Phe(6.52)] and allows a tighter binding of the ligands when compared with the rat receptor.