We have previously demonstrated that interleukin 3 (IL-3) has a neurotrophic effect on central cholinergic neurons and have demonstrated the presence of IL-3 receptor (IL-3R)β subunits in septal cholinergic neurons by reverse-transcribed polymerase chain reaction (RT-PCR) and immunohistochemistry. In order to confirm that the expressed IL-3R is functional, we conducted experiments to show an a subunit of IL-3R. The α subunit was clearly demonstrated by RT-PCR in the central cholinergic neuronal hybrid cell line SN6, but not in its mother cell line N18TG2, and the expression was slightly upregulated after IL-3 treatment. Choline acetyltransferase and vesicular acetylcholine transporter mRNAs were significantly increased in SN6 after treatment with IL-3. Immunohistochemically, IL-3Rα-positive cells were mainly present in the medial septal and basal forebrain region, and the stained cells were similar to choline acetyltransferase-positive cells in shape and distribution. The IL-3Rα-positive cells slightly increased two days after fimbria-fornix transection and decreased seven days after. These findings suggest that functional IL-3 receptors are expressed in the central cholinergic neurons and contribute to some physiological roles such as the differentiation and maintenance of these neurons.