In this report the normal dendritic organization and fine structure of identified septohippocampal projection neurons is described as a prerequisite for a time course analysis of retrograde changes in these neurons following axotomy (see Naumann et al., J. Comp. Neurol. 325:219-242, 1992). Septohippocampal projection neurons were retrogradely labeled by injection of the fluorescent tracer Fluoro-Gold into the hippocampus. Next, retrogradely labeled cells in Vibratome sections of the medial septum/diagonal band complex were intracellularly stained with the fluorescent dye Lucifer Yellow (LY). Photooxidation of LY resulted in a stable electron-dense reaction product, which allowed us to study these double-labeled neurons by electron microscopy. Another series of sections containing retrogradely labeled neurons were immunostained for choline acetyltransferase (ChAT) or parvalbumin (PARV). In this way the fine structure of two different chemically characterized subpopulations of septohippocampal neurons could be compared with that of the LY-injected neurons. Intracellular filling of retrogradely labeled neurons with LY stained the cell body and the entire dendritic arbor. Essentially, three classes of neurons could be distinguished, i.e., bipolar cells, multipolar neurons, and an intermediate group. All these neurons displayed smooth, often varicose dendrites lacking spines. Mainly located close to the midline, there was a group of cells with only very few if any LY-stained dendrites. In the electron microscope, the double-labeled neurons were easily identified by numerous electron-dense lysosomes associated with transported Fluoro-Gold and the diffuse reaction product resulting from photooxidation. They displayed fine-structural characteristics as previously described for cholinergic neurons. In fact, our fine-structural analysis of ChAT-positive Fluoro-Gold-labeled neurons, but also of back-filled PARV-positive cells, gave very similar results. All these neurons had infolded nuclei, abundant cytoplasmic organelles, and a few axosomatic synapses. Thus, a plain electron microscopic study does not allow one to distinguish between subpopulations of septohippocampal projection neurons.