The cloned 5-HT3 receptor from NCB-20 neuroblastoma cells was expressed in Xenopus oocytes and the effect of the endogenous cannabinoid ligand, anandamide, was investigated on the function of this receptor. The oocytes expressing the cloned 5-HT3 receptors were voltage-clamped at -70 mV. Anandamide, at the concentration range of 0.1-100 microM, reversibly inhibited 1 microM 5-HT induced currents. The inhibition of 5-HT induced currents by anandamide was concentration-dependent with an EC50 of 3.7 microM and slope value of 0.94. This inhibitory effect was not dependent on the membrane potential and anandamide did not have an effect on the reversal potential of 5-HT-induced currents. In the presence of 10 microM anandamide, the maximum 5-HT-induced response was also inhibited and the respective EC50 values were 3.4 microM and 3.1 microM in the absence and presence of anandamide, indicating that anandamide acts as a noncompetitive antagonist on 5-HT3 receptors. CB1 receptor antagonist SR-141716A (1 microM) and pertussis toxin (5 microg/ml) did not cause a significant change on the inhibition of 5-HT responses by anandamide. The effect of anandamide was not changed by preincubating the oocytes with 0.2 mM 8-Br-cAMP, a membrane-permeable analog of cAMP, or Sp-cAMPS (0.1 mM), a membrane-permeable protein kinase A activator. These results suggest that the effect of anandamide is independent of the activation of cAMP pathway and not mediated by the activation of PTX sensitive G-proteins. In conclusion, we demonstrated that the endogenous cannabinoid anandamide inhibits the function of 5-HT3 receptors expressed in Xenopus oocytes in a cannabinoid-receptor independent and noncompetitive manner.