Single 5-HT3 receptor-gated ion channels were recorded in cell-attached and excised membrane patches of mouse N1E-115 neuroblastoma cells. In cell-attached patches 5-HT3 receptor-gated ion channels show distinct conductance levels of 27, 18, 11 and 6 pS. Patch excision, buffering of intracellular Ca2+ by BAPTA and inhibition of protein kinase activity by staurosporine increase the probability of occurrence of the 6 pS level and decrease that of the 27 pS level. Conversely, patch cramming and stimulation of protein kinase activity by the phorbol ester PMA enhance the probability of occurrence of the 27 pS level and decrease that of low conductance levels. We conclude that phosphorylation controls the conductance of 5-HT3 receptor ligand-gated ion channels. Control of channel conductance may prove an additional mechanism in the modulation of synaptic efficacy.