For many years neuroanatomical research has focused on the tracing of connections between populations of neurons in the central nervous system. With the development of immunocytochemical procedures employing antibodies against transmitters, transmitter-synthesizing enzymes and various neuropeptides, it has become possible to study the chemical characteristics of nerve cells. By combining tracer techniques and immunocytochemical approaches the transmitter identity of a projection can be determined. Moreover, electron microscopic double-labeling procedures allow the identification of the target cells of immunocytochemically characterized presynaptic terminals. This way, by the immunocytochemical identification of transmitters or neuropeptides in defined projection neurons, a more functional interpretation of neuroanatomical data has become possible. In the present report, these combinations of techniques are demonstrated by describing recent studies of cholinergic septohippocampal projection neurons.