The I.T50 method consists of determining the charge required to obtain a 50% depression of firing activity of neurons recorded extracellularly with microiontophoretic applications of inhibitory agents. This method has been used successfully to detect modification of neuronal responsiveness, but the limits of its validity had never been determined. In the present study, it was found that the use of microiontophoretic currents greater than 3 nA yielded consistent I.T50 values when serotonin (5-HT) was applied to rat hippocampal pyramidal neurons. The departure from linearity of I.T50 values measured from applications carried out with a very low current (0.5 nA) of 5-HT is probably due to the relatively important contribution of the leak when a minimal ejecting current is used. The responsiveness to 5-HT was not altered by the activation of the recorded neuron produced by acetylcholine.