To date, acetylcholinesterase (AChE) inhibitors have been clinically effective drugs for the palliative treatment of Alzheimer's disease, but their clinical efficacy is limited, mainly due to their adverse effects on peripheral organs. Since patients of Alzheimer's disease often exhibit depression as well as memory impairment, dual inhibitors of AChE and serotonin transporter (SERT) would be a better therapeutic method. Anti-depressive effects based on SERT inhibition would reduce the dose-related side effects of AChE inhibitors. Such dual inhibitors were designed by the hybridization of rivastigmine and fluoxetine based on a hypothetical model of the AChE active site. Various derivatives were synthesized and evaluated for their in vitro inhibition, and then (S)-5j (RS-1259), which possessed balanced inhibitory activities of AChE (IC(50)=101 nM) and SERT (IC(50)=42 nM), was successfully obtained. An ex vivo experiment in mice indicated that (S)-5j (RS-1259) simultaneously inhibited AChE and SERT in the brain following an oral administration. The simultaneous elevation of extracellular levels of acetylcholine and serotonin in the rat hippocampus was actually confirmed by microdialysis.