Intracellular recordings were made from fetal mouse spinal cord neurons in primary culture. One type of neuron, with large somata (40–50 μm diameter) and thick neuntes exhibited endogenous bursting or beating pacemaker electrical activity. Noradrenaline depolarized this type of neuron by decreasing an M-like conductance. Micropressure application of serotonin (10 −5 M in the delivery pipette) onto the surface of pacemaker neurons evoked a depolarization of the membrane potential in a dose-dependent manner with an increased input resistance. No such response was observed with other types of spinal cord neurons in culture. The response to serotonin was partially voltage-dependent. The serotonin-induced depolarization reversed at holding potential close to − 100 mV. However, the input resistance variation evoked by serotonin increased exponentially when membrane potential was depolarized. The reversal potential was modified by increasing extracellular K + concentration and it was unaltered by increasing the intracellular Cl − concentration. The decrease in K + conductance induced by serotonin was not suppressed by the application of tetraethylammonium (50 mM) or 4-aminopyridine (10 mM). Furthermore, application of Ba 2+ (6mM) or Cd 2+ (0.1 mM) had no effect on this response, suggesting that the depolarization evoked by serotonin application was not calcium-dependent. The serotonin evoked increase in input resistance was mediated by activation of a 5-HT 1A-like receptor site. Spiperone, a 5-HT 1A antagonist reversibly blocked the response. Methiothepin, a 5-HT 1-5-HT 2 antagonist (10 −3 M); cocaine, a 5-HT 3 antagonist (10 −3 M); ketanserine, a 5-HT 2 antagonist (10 −3 M); and prazosin, an α 1, antagonist (10 −3 M) had no effect. Pacemaker neurons possessed both serotonin and noradrenaline receptor sites. When serotonin and noradrenaline were applied separately, serotonin evoked a response of longer duration than those evoked by noradrenaline. However, simultaneous application of noradrenaline and serotonin did not evoke summation of the responses suggesting that serotonin and noradrenaline act on the same K + channel and/or that serotonin and noradrenaline receptor sites were coupled, possibly by second messenger mechanisms.