The cerebral ganglia neurons of Aplysia californica are involved in the development and modulation of many behaviors. The medially located F cluster has been characterized using morphological, electrophysiological and biochemical techniques and contains at least three previously uncharacterized neuronal population. As the three subtypes are located in three distinct layers, they are designated as top, middle, and bottom layer F-cluster neurons (CFT, CFM, and CFB). The CFT cells are large (92 +/- 25 microm), white, nonuniformly shaped, and located partially in the sheath surrounding the ganglion. These neurons exhibit weak electrical coupling, the presence of synchronized spontaneous changes in membrane potential, and a generalized inhibitory input upon electrical stimulation of the anterior tentacular (AT) nerve. Similar to the CFT neurons, the CFM neurons (46 +/- 12 microm) are mainly silent but do not show electrical coupling or synchronized changes in membrane potential. Unlike the CFT neurons, the CFM neurons exhibit weak action potential broadening during constant current injection. Comparison of the peptide profiles of CFT, CFM, and CFB (10-30 microm) neurons using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry demonstrates distinct peptide molecular weights for each neuronal subtype with the masses of these peptides not matching any previously characterized peptides from A. californica. The mass spectra obtained from the AT nerve are similar to the CFT neuron mass spectra, while upper labial nerve contains many peptides observed in the CFM neurons located in nongranular neuron region.