Neurons in the CNS generally receive inputs form multiple afferent sources. These afferent systems seldom all use the same neurotransmitter, so most central neurons are required to express multiple neurotransmitter receptors. This work addresses the issue of how multiple neurotransmitter receptors are regulated on the surface of individual neurons. We made whole-cell voltage-clamp recordings from identified chick sympathetic preganglionic neurons (SPNs) in dissociated cell cultures. The neurons were derived from stage 30-31 (7 d) chick embryos and were studied within the first week in vitro. We found that by 1 week in vitro, most SPNs responded to the application of GABA, glycine, and glutamate. The responses of SPNs to the amino acid neurotransmitters were similar to the responses of other CNS neurons to these 3 substances. SPNs became sensitive to these substances at different times in culture. At 1 d in vitro, most cells already responded to GABA, and about half of the cells also responded to glycine and kainate. In contrast, responses to NMDA and quisqualate were usually not seen until day 3-4 in vitro. Although there was a general trend for the amplitude of the responses of SPNs to each of the neurotransmitters to increase with time in vitro, there was an immense amount of cell-to-cell variability. By measuring the amplitudes of the responses of a series of SPNs to all 3 transmitters, we were able to test whether a common regulatory mechanism governed the level of responsiveness of SPNs to all 3 amino acid transmitters. We found no correlation between cells in the amplitudes of their responses to the 3 transmitters. Both the differences in time course of appearance of responsiveness and the lack of correlation in the amplitude of responses suggest that the multiple receptors on the surface of SPNs in vitro are independently regulated.