The second β-like subunit (SBD) is a putative structural subunit of Drosophila melanogaster nicotinic acetylcholine receptors (nAChRs). Here we have produced specific antibodies against SBD to study, which other nAChR subunits can co-assemble with SBD in receptor complexes of the Drosophila nervous system. Immunohistochemical studies in the adult optic lobe revealed that SBD has a distribution similar to␣that of the α-subunit ALS in the synaptic neuropil. The subunits ALS, Dα2 and SBD can be co-purified by α-bungarotoxin affinity chromatography. Moreover, anti-SBD antibodies co-precipitate ALS and Dα2 and, vice versa, ALS and Dα2 antibodies co-immunoprecipitate SBD protein. Two-step immunoaffinity chromatography with immobilized antibodies against ALS and Dα2 revealed the existence of nAChR complexes that include ALS, Dα2 and SBD as integral components. Interestingly, the genes encoding these three subunits appear to be directly linked in the Drosophila genome at region 96 A of the third chromosome. In addition, SBD appears to be a component of a different receptor complex, which includes the ARD protein as an additional β-subunit, but neither ALS nor Dα2 nor the third α-subunit Dα3. These findings suggest a considerable complexity of the Drosophila nicotinic receptor system.