Nervous and immune systems maintain a bidirectional communication, expressing receptors for neurotransmitters and cytokines. Despite being well established in mammals, this has been poorly described in lower vertebrates as fishes. Experimental evidence shows that the neurotransmitter acetylcholine (ACh) regulates the immune response. In this research, we evaluated mRNA levels of muscarinic acetylcholine receptor (mAChR) in spleen mononuclear cells of Nile tilapia (Oreochromis niloticus) and compared the expression levels of immune cells with the brain. The mAChR subtypes (M2-M5A) were detected in both tissues, but mAChRs mRNA levels were higher in immune cells. This data have a potential use in biomedical and comparative immunology fields. Display omitted •Cholinergic receptors are present on fish leukocytes.•Brain and spleen mononuclear cells express the muscarinic acetylcholine receptor subtypes M2, M3, M4, and M5A.•The mRNA levels of all the evaluated mAChR subtypes were higher on spleen mononuclear cells than the brain.