The influence of non-neuronal cells and interneurons on the morphological development of chick sympathetic preganglionic neurons (SPNs) and on the responsiveness of these neurons to the neurotransmitters GABA, glycine, and glutamate was studied. SPNs were retrogradely labeled with the fluorescent dyes dil and diO, then separated from spinal-cord non-neuronal cells and interneurons by fluorescence-activated cell sorting. SPNs were grown in culture, either alone or in coculture with non-neuronal cells alone, with interneurons alone, or with both of these cell types (control cultures). The responsiveness of SPNs to neurotransmitters was assessed by whole-cell recording, while cell morphology was assessed after intracellular staining with 6-carboxyfluorescein. Cell size and morphology were affected by non-neuronal cells. In the absence of non-neuronal cells, SPNs had smaller cell bodies and fewer major processes, whether or not interneurons were present. In contrast, responses to the 3 neurotransmitters were affected by both non-neuronal cells and interneurons, but in ways that differed slightly for each transmitter. In the absence of both non-neuronal cells and interneurons, responses to all 3 transmitters were much smaller than in control cultures, with responses to glutamate most profoundly affected. The addition of either non-neuronal cells or interneurons slightly increased the amplitude of SPN responses to glutamate, but the level of responsiveness with either cell type alone was much lower than for SPNs grown in the presence of both cell types. The addition of interneurons also slightly increased the responsiveness of SPNs to GABA, but non-neuronal cells alone had no significant effect on the responses of SPNs to GABA. Finally, the glycine responsiveness of SPNs was raised to control levels when either non-neuronal cells or interneurons were added. These experiments demonstrate that, though interneurons can have a significant inductive effect on the responses of SPNs to neurotransmitters, not all of the changes in neurotransmitter responsiveness can be related to the formation of functional synapses.