The wild-type and a mutant 5-HT3 receptor were expressed in Xenopus oocytes to further explore how the rate of onset of desensitization of the 5-HT3 receptor was dependent on membrane voltage and primary structure of the channel. The rapid application of serotonin (5-HT; 50 microM) in a Ca(2+)-containing (1.8 mM) bathing solution elicited inward currents that decayed rapidly and with a biphasic time course in most cases. For oocytes expressing the wild-type 5-HT3 receptor and held at a potential of -90 mV, the value of the fast time constant of decay (tau fast) was 0.79 +/- 0.3 s (n = 7), while tau slow was 16 +/- 3 s; the area of the decay phase contributed by tau fast (i.e., Afast) was 50 +/- 4% and Aslow was 38 +/- 5%, with a remainder (i.e., non-desensitizing component) of 12%. The kinetics of the decay phase were strongly voltage-dependent. At a holding potential of -30 mV, the desensitization decay phase was now monophasic, with a time constant of 14.0 +/- 3.1 s (n = 4). Mutating the leucine at position 286 of the wild-type 5-HT3 receptor to threonine (i.e., mutant L286T) resulted in desensitization kinetics that were biphasic at all membrane potentials tested and with a rate of decay that was not voltage dependent. Therefore, desensitization is a multifaceted and complex process, whose rate of onset depends in part to membrane voltage and primary structure of the ion channel.