Serotonin 5-HT2 receptor-mediated intracellular Ca2+ mobilization was investigated in rat glioma C6BU-1 cells. The receptors became desensitized after previous exposure to 5-HT in a time- and concentration-dependent manner. The desensitization of 5-HT2 receptor-mediated intracellular signaling appeared to be homologous because previous exposure to 5-HT did not alter the response to other transmitters such as thrombin or isoproterenol and because previous exposure to thrombin or isoproterenol did not diminish the response to 5-HT. The desensitization induced by pretreatment with 5-HT was potently prevented by the naphthalenesulfonamide derivative W-7, a calmodulin antagonist, when it was cosupplied with 5-HT. Furthermore, the preventive effect of W-7 was greater than that of W-5, a weak analogue of W-7, and than that of H-7, a nonselective inhibitor of protein kinases. These results suggest that 5-HT2 receptor-mediated Ca2+ mobilization can be desensitized homologously after prolonged exposure to 5-HT in a calmodulin-dependent manner in rat glioma C6BU-1 cells.