Neuroblastoma x glioma hybrid NG108-15 cells and mouse neuroblastoma N18TG-2 and N1E-115 cells were transiently transfected with the sense cDNA coding for rat choline acetyltransferase (ChAT). All transfected cell lines showed a high level of ChAT activity. ACh secretion was monitored by recording miniature end-plate potentials (MEPPs) in striated muscle cells that had been co-cultured with transfected cells. The number of muscle cells with synaptic responses and the MEPP frequency were higher in co-culture with transfected NG108-15 cells than with control or mock cells. No synaptic response was detected in muscle cells co-cultured with transfected N18TG-2 or N1E-115 cells. The results show that ACh secretion into the synaptic cleft was enhanced due to ChAT overexpression in NG108-15 hybrid cells but not in neuroblastoma cells.