Immunolabeling of β2 and α4 subunits was quantitated in brain sections (14 μm) using [125I]mAb 270 and [125I]mAb 299, respectively. Specificity was demonstrated by signal loss in β2−/− and α4−/− brain sections, respectively. Even mild paraformaldehyde fixation severely affected immunolabeling, so this study used unfixed sections. Immunolabeling autoradiography was used to map and quantitate the effects of β2 and α4 subunit-null mutations on their putative partner subunits' protein expression. [125I]mAb 299 labeling was nearly eliminated in β2−/− sections, although dorsal interpeduncular nucleus (IPN) retained a faint signal. Therefore, α4 subunit expression is almost universally β2-dependent. In contrast, α4-null mutation effects on [125I]mAb 270 immunolabeling varied widely among brain regions. In corticothalamic regions, [125I]mAb 270 labeling was eliminated. However, in habenulopeduncular regions, α4 genotype had no effect. Other (predominantly dopaminergic and optic tract) nuclei also retained reduced [125I]mAb 270 labeling in α4−/− sections. Thus, although most β2 subunit protein expression is α4-dependent, this dependence is not universal. Presumably, residual β2 subunits are found in non-α4* subtypes. Together, these results show that immunolabeling is applicable to reliable, quantitative investigations of neuronal nAChRs, and that subunit-null mutants can be appropriate controls for such experiments. In situ mRNA hybridization was also performed to determine if altered mRNA transcription mediated the interdependence of α4 and β2 subunit expression. α4-Null mutation did not affect β2 mRNA expression, nor did β2 genotype affect α4 mRNA expression. Consequently, it seems that the two subunits' effects on each other's expression are mediated at the protein, rather than gene expression level. J. Comp. Neurol. 499:1016–1038, 2006. © 2006 Wiley-Liss, Inc.