In this study, the relationship between substance P-immunoreactive boutons and antidromically activated sympathetic preganglionic neurons was examined by light and electron microscopy. Sympathetic preganglionic neurons in the T2-T4 spinal segments of the cat were identified by intracellular recording and antidromic activation from the corresponding white ramus. Neurons were filled with lucifer yellow and then stained to reveal, simultaneously, substance P and lucifer yellow immunoreactivity. All of the neurons examined with the light microscope (n = 13) received appositions from substance P-immunoreactive boutons. Appositions were found on all parts of the neuron, including the somata, dendrites, and axon initial segment. In most cases (11/13) few close appositions were seen; however, two neurons received large numbers of appositions from substance P-immunoreactive boutons. On one neuron, 16 substance P-immunoreactive varicosities that were identified as being closely apposed at the light microscope level were serially sectioned and examined with the electron microscope. Of these 16 varicosities, eight either directly contacted the neuron or formed morphologically identifiable synapses. The remaining eight varicosities were separated from the neuron by thin glial processes. Two other sympathetic preganglionic neurons that were examined ultrastructurally also received substance P-immunoreactive synapses and close contacts. These findings suggest that substance P-containing nerve fibres could affect all sympathetic preganglionic neurons but are likely to be important in regulating the activity of only a small proportion of these neurons.