1. The time course of macroscopic current responses of homomeric murine serotonin 5-HT3A receptors was studied in whole cells and excised membrane patches under voltage clamp in response to rapid application of serotonin. 2. Serotonin activated whole cell currents with an EC(50) value for the peak response of 2 microM and a Hill slope of 3.0 (n = 12), suggesting that the binding of at least three agonist molecules is required to open the channel. 3. Homomeric 5-HT3A receptors in excised membrane patches had a slow activation time course (mean +/- S.E.M. 10-90 % rise time 12.5 +/- 1.6 ms; n = 9 patches) for 100 microM serotonin. The apparent activation rate was estimated by fitting an exponential function to the rising phase of responses to supramaximal serotonin to be 136 s(-1). 4. The 5-HT3A receptor response to 100 microM serotonin in outside-out patches (n = 19) and whole cells (n = 41) desensitized with a variable rate that accelerated throughout the experiment. The time course for desensitization was described by two exponential components (for patches tau(slow) 1006 +/- 139 ms, amplitude 31 %; tau(fast) 176 +/- 25 ms, amplitude 69 %). 5. Deactivation of the response following serotonin removal from excised membrane patches (n = 8) and whole cells (n = 29) was described by a dual exponential time course with time constants similar to those for desensitization (for patches tau(slow) 838 +/- 217 ms, 55 % amplitude; tau(fast) 213 +/- 44 ms, 45 % amplitude). 6. In most patches (6 of 8), the deactivation time course in response to a brief 1-5 ms pulse of serotonin was similar to or slower than desensitization. This suggests that the continued presence of agonist can induce desensitization with a similar or more rapid time course than agonist unbinding. The difference between the time course for deactivation and desensitization was voltage independent over the range -100 to -40 mV in patches (n = 4) and -100 to +50 mV in whole cells (n = 4), suggesting desensitization of these receptors in the presence of serotonin does not reflect a voltage-dependent block of the channel by agonist. 7. Simultaneously fitting the macroscopic 5-HT3A receptor responses in patches to submaximal (2 microM) and maximal (100 microM) concentrations of serotonin to a variety of state models suggests that homomeric 5-HT3A receptors require the binding of three agonists to open and possess a peak open probability greater than 0.8. Our modelling also suggests that channel open probability varies with the number of serotonin molecules bound to the receptor, with a reduced open probability for fully liganded receptors. Increasing the desensitization rate constants in this model can generate desensitization that is more rapid than deactivation, as observed in a subpopulation of our patches.