Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of anti‐receptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiological findings. In this context, nicotinic acetylcholine receptor subunit‐deficient mice should be ideal tools for testing the specificity of subunit‐directed antibodies. Here, we used standard protocols for immunohistochemistry and western blotting to examine the antibodies raised against the α3‐, α4‐, α7‐, β2‐, and β4‐nicotinic acetylcholine receptor subunits on brain tissues of the respective knock‐out mice. Unexpectedly, for each of the antibodies tested, immunoreactivity was the same in wild‐type and knock‐out mice. These data imply that, under commonly used conditions, these antibodies are not suited for immunolocalization. Thus, particular caution should be exerted with regards to the experimental approach used to visualize nicotinic acetylcholine receptors in the brain.