Nicotinic acetylcholine receptors control survival, proliferation and cytokine release in non-excitable cells. Previously we reported that α7 nicotinic receptors were present in the outer membranes of mouse liver mitochondria to regulate mitochondrial pore formation and cytochrome c release. Here we used a wide spectrum of nicotinic receptor subunit-specific antibodies to show that mitochondria express several nicotinic receptor subtypes in a tissue-specific manner: brain and liver mitochondria contain α7β2, α4β2 and less α3β2 nicotinic receptors, while mitochondria from the lung express preferentially α3β4 receptor subtype; all of them are non-covalently connected to voltage-dependent anion channels and control cytochrome c release. By using selective ligands of different nicotinic receptor subtypes (acetylcholine (1 μM) or dihydro-β-erythroidine (1 μM) for α4β2), conotoxin MII (1 nM) for α3β2, MLA (50 nM) for α7β2 and acetylcholine (10 μM) for all subtypes) and apoptogenic agents triggering different mitochondrial signaling pathways (1 μM wortmannin, 90 μM Ca2+or 0.5 mM H2O2) it was found that α7β2 receptors affect mainly PI3K/Akt pathway, while α3β2 and α4β2 nAChRs also significantly influence CaKMII- and Src-dependent pathways. It is concluded that cholinergic regulation in mitochondria is realized through multiple nicotinic receptor subtypes, which control various pathways inducing mitochondrial type of apoptosis. © 2014 Elsevier Ltd.