The effects of delta9-tetrahydrocannabinol and 2-arachidonoylglycerol on the intracellular free Ca2+ concentration ([Ca2+]i) in NG108-15 cells were examined in detail. We found that delta9-tetrahydrocannabinol induces a rapid, modest increase in [Ca2+]i. The response was detectable with 3 nM delta9-tetrahydrocannabinol. We also found that very low concentrations of 2-arachidonoylglycerol elicit a rapid, more prominent increase in [Ca2+]i. Such a response was observed not only in NG108-15 cells but also in N18TG2 cells. The response induced by 2-arachidonoylglycerol in either NG108-15 cells or N18TG2 cells was abolished by pretreatment of the cells with a cannabinoid CB1 receptor specific antagonist, SR141716A, suggesting that 2-arachidonoylglycerol interacts with the CB1 receptor to induce the response. The results of an experiment involving a phospholipase C inhibitor suggested that phospholipase C is involved in the rapid increase in [Ca2+]i induced by 2-arachidonoylglycerol. We also found that 1(3)-arachidonoylglycerol exhibits similar activity to that of 2-arachidonoylglycerol, although its activity at low concentrations was somewhat weak compared with that of 2-arachidonoylglycerol. We further confirmed that several structural analogues of 2-arachidonoylglycerol were less active compared with 2-arachidonoylglycerol. These results suggest that the structure of 2-arachidonoylglycerol is strictly recognized by the CB1 receptor, which raises the possibility that the CB1 receptor is originally a 2-arachidonoylglycerol receptor.