Within the chick central nervous system, expression of the β3nicotinic acetylcholine receptor gene is restricted to a subset of retinal neurons, the majority of which are ganglion cells. Transient transfection in retinal neurons and in neural and non-neural cells from other regions of the click embryo allowed the indentification of the cis-regulatory domain of the β3 gene. Within this domain, a 75-base pair fragment located immediately upstream of the transcription start site suffices to reproduce the neuron-specific expression pattern of β3. This fragment encompasses an E-box and a CAAT box, both of which are shown to be key positive regulatory elements of the β3 promoter. Co-transfection experiments into retinal, telencephalic, and tectal neurons with plasmid reporters of β3 promoter activity and a number of vectors expressing different neuronal (ASH-1, NeuroM, NeuroD, CTF-4) and non-neuronal (MyoD) basic helix-loop-helix transcription factors indicate that the cis-regulatory domain of β3 has the remarkable property of discriminating accurately between related members of the basic helix-loop-helix protein family. The sequence located immediately 3' of the E-box participates in this selection, and the E-box acts in concert with the nearby CAAT box.