The novel antidepressant, agomelatine, behaves as an agonist at melatonergic receptors, and as an antagonist at edited, human serotonin2C(VSV) receptors [h5-HT2C(VSV)Rs]. However, its actions at constitutively active 5-HT2CRs have yet to be characterized, an issue addressed herein. At unedited h5-HT2C(INI)Rs expressed in HEK-293 cells, 5-HT enhanced [35S]GTPγS binding to Gαq, whereas the inverse agonists SB206,553 and S32006 inhibited binding and, by analogy to the neutral antagonist, SB242,084, agomelatine exerted no effect alone. Mirroring these observations, 5-HT stimulated, whereas SB206,553 and S32006 inhibited, [3H]inositol phosphate formation. Both the agonist actions of 5-HT and the inverse agonist actions of SB206,553 and S32006 were abolished by agomelatine and SB242,084. As demonstrated by bioluminescence resonance energy transfer, 5-HT enhanced, whereas SB206,553 and S32006 decreased, association of ‘h5-HT2C(INI)-Rluc-tagged’ receptors with yellow-fluorescence-protein-coupled β-arrestin2. These actions of 5-HT, SB206,553 and S32006 were prevented by agomelatine and SB242,084 were ineffective alone. As shown by ELISA and confocal microscopy, prolonged (18 h) exposure to SB206,553 or S32006 enhanced cell surface expression of N-terminal Flag-tagged h5-HT2C(INI)Rs: these effects were blocked by agomelatine and SB242,084, which were inactive alone. Finally, following pre-exposure to SB206,553 or S32006 for 18 h, 5-HT triggered 5-HT2CR-mediated elevations in cytosolic Ca2+ in primary cultures of mice cortical neurons. Agomelatine and SB242,084, inactive alone, prevented these actions of SB206,553 and S32006. In conclusion, agomelatine behaves as a neutral antagonist at constitutively active h5-HT2C(INI)Rs and native, cortical 5-HT2CRs. It will be of interest to determine whether the neutral antagonist properties of agomelatine are related to its favourable clinical profile of antidepressant properties with few side-effects and no discontinuation syndrome.