The diversity of nicotinic ACh receptor (AChR) expression in the chick lateral spiriform nucleus (SpL) was assessed using subunit-specific monoclonal antibodies (mAbs) and laser scanning confocal microscopy. The late embryonic SpL was immunoreactive for mAbs against the alpha 2, alpha 5, alpha 7, alpha 8, and beta 2 AChR subunits. Distinct neuronal cell classes were determined using pair-wise staining of mAbs. Approximately 90% of the neurons in the SpL contained both alpha 5-like immunoreactivity (LI) and beta 2-LI, with no neurons having only one of these subunit-LIs. Approximately 70% of the neurons contained alpha 2- LI. All alpha 2-LI neurons contained alpha 5/beta 2-LI; thus, neurons having alpha 2-LI are a subset of those having alpha 5- and beta 2-LI. Fewer neurons, approximately 20%, contained alpha 7-LI. A subset of alpha 7-positive neurons were immunoreactive for other subunits; for example, some alpha 7-positive neurons also contained alpha 2-LI. Fewer than 15% of the neurons contained alpha 8-LI. Some of the alpha 8-LI- containing neurons contained alpha 7-LI. The 14 week post-hatch SpL resembles the late embryonic nucleus in the percentage of neurons immunoreactive for alpha 2, alpha 5, alpha 7, alpha 8, and beta 2 AChR subunits, and in the presence of multiple classes based on AChR subunit immunoreactivity. In addition, alpha 4-LI was found in about 20% of the 14 week SpL neurons. Double-label immunofluorescence experiments with mAbs to AChRs and to synaptic vesicle antigens showed that most clusters of alpha 5-LI and beta 2-LI are extrasynaptic. The pronounced diversity of AChR subunit expression and the extrasynaptic location of AChR-LI suggest that AChR-like molecules in the SpL do not function solely to respond to transmitter focally released from presynaptic terminals.