The ability of differentiated neurons to secrete false transmitters in response to depolarization was examined by loading exogenous transmitters into the neuronal cytoplasm with a whole-cell recording pipette. We found that within minutes following loading of exogenous glutamate into the cytoplasm of cholinergic Xenopus spinal neurons, depolarization-evoked glutamate secretion could be detected by an acutely dissociated hippocampal pyramidal neuron manipulated into contact with the spinal neuron. Similarly, when ACh was loaded into a glutamatergic hippocampal neuron, evoked ACh secretion could be detected by a myocyte. The evoked secretion of the false transmitter was Ca2+ dependent and appeared to be vesicular in nature. These results suggest that differentiated neurons are capable of packaging and secreting multiple nonpeptide transmitters, provided that sufficient concentrations of the transmitters are available in the cytoplasm.