In the present paper, we describe affinities to 5-HT1A and 5-HT2A receptors of several new 1,2,4-trisubstituted piperazine derivatives. The affinities were compared with those described earlier for 1,4-disubstituted piperazines and the influence of the third (methyl) substituent on the affinity to both receptors is discussed. The difference between two- and three-substituted derivatives was rationalised in terms of molecular modelling of the respective ligand-receptor complexes. Additionally, the functional activity of some 1,2,4-trisubstituted piperazines for 5-HT1A receptor was examined in behavioural and biochemical models. The obtained results have shown that some trisubstituted compounds exhibited a higher affinity to 5-HT2A receptors than their respective disubstituted analogues (with the affinity to 5-HT1A receptors remaining the same or somewhat improving). The molecular dynamics simulations suggested that the presence of the third substituent in the piperazine ring of those compounds may induce stabilising effect on the ligand-receptor complexes. The results of the in vivo studies have shown that some of the examined trisubstituted piperazines (10-13, 16, 17) exhibited properties of postsynaptic 5-HT1A partial agonists. Moreover, compounds 13 and 16 exhibited features of 5-HT1A presynaptic agonists in in vitro test, and compound 16 also in in vivo tests.