The serotonin 6 receptor (5-HT 6 R) is one of the most recently cloned receptors among the known serotonin receptors. Because it is abundantly distributed in the limbic region and is involved in neurological disorders, it has generated much interest as a drug discovery and a biological research target. However, several groups recently reported conflicting results for 5-HT 6 R ligands in animal studies. Herein, we investigated in vitro effects of 5-HT 6 R ligands in cultured neurons and mammalian cell lines to explain inconsistencies in the results of in vivo studies. When examined the effects of EMD386088, a 5-HT 6 R agonist, on cell viability, we found that EMD386088 potentiated cell death in cultured neuronal, native HEK293, and HEK/HA-5-HT 6 R cells. The results demonstrated that EMD386088 induces its cytotoxic effects, regardless of the presence of 5-HT 6 Rs, by the downregulation of ERK1/2 activities. Furthermore, we studied the 5-HT 6 R-independent effects of SB258585, a specific 5-HT 6 R antagonist. SB258585 potentiated cell death and induced an increase in [Ca 2+ ] i , whereas EMD386088 or 5-HT did not affect [Ca 2+ ] i . Because EMD386088 and SB258585 have been intensively used as 5-HT 6 R ligands in in vitro and in vivo studies, our findings suggest that these compounds should be used with caution in cell-based studies as well as behavioral studies.