Abstract: Neuronal nicotinic acetylcholine receptors are differentially sensitive to blockade by the competitive antagonist dihydro-β-erythroidine. Both α and β subunits participate in determining sensitivity to this antagonist. The α subunit contribution to dihydro-β-erythroidine sensitivity is illustrated by comparing the α4β4 receptor and the α3β4 receptor, which differ in sensitivity to dihydro-β-erythroidine by ∼120-fold. IC50 values for blocking α4β4 and α3β4, responding to EC20 concentrations of acetylcholine, were 0.19 ± 0.06 and 23.1 ± 10.2 µM, respectively. To map the sequence segments responsible for this difference, we constructed a series of chimeric α subunits containing portions of the α4 and α3 subunits. These chimeras were coexpressed with β4, allowing pharmacological characterization. We found determinants of dihydro-β-erythroidine sensitivity to be distributed throughout the N-terminal extracellular domain of the α subunit. These determinants were localized to sequence segments 1–94, 94–152, and 195–215. Loss of determinants within segment 1–94 had the largest effect, decreasing dihydro-β-erythroidine sensitivity by 4.3-fold.