Rebaudioside A (RebA) is a steviol glycoside used for production of sweeteners. It was shown that the glycosides affect memory and learning processes. The aim of the study was to investigate neurons immunoreactive for acetylcholinesterase (AChE) and M1 muscarinic receptors (mAChRs-M1) of the hippocampal CA1 and CA3 fields and striatal caudateputamen (CP) and globus pallidus (GP) in rats receiving RebA. RebA was administrated to adult rats for 45 days in dilutions of 1 mg and 2 mg RebA/ml water. Indirect peroxidase-antiperoxidase immunohistochemical reaction was conducted on frontal sections containing the hippocampus and striatum with use of antibodies against AChE and mAChRs-M1. Immunoreactive for the studied proteins neurons were morphologically and morphometrically assessed in hippocampal CA1 and CA3 fields and in CP and GP. Microscopic observations did not reveal significant changes in morphology of immunoreactive neurons, which suggests no neurotoxic effect of the studied glycoside on these cells. Morphometric analyses revealed an increase in the density of AChE and mAChRs-M1 immunoreactive neurons. A decrease in reaction intensity of AChE-positive neurons was also demonstrated in the hippocampal CA1 field and in GP. In contrast, an increase in reaction intensity of mAChRs-M1-positive neurons was found in CA1, CA3 fields and in CP and GP. The results of our preliminary studies indicate that RebA administrated to rats has an impact on cholinergic neurons in the studied area. The results suggest a possible increase in the activity of the cholinergic system, responsible for memory and learning processes, after administration of RebA.