AIMS: This study aimed to investigate the influence of physiological ions on the transport of acetylcholine which is catalyzed by the recombinant human Organic Cation Transporter Novel 1 (hOCTN1), thus being involved in the function of the non neuronal cholinergic system. MAIN METHODS: The experimental model of proteoliposomes reconstituted with the hOCTN1 transporter obtained by over-expression in E. coli has been used. Uptake and efflux of [(3)H]acetylcholine in the proteoliposome system have been followed in the presence of different cations, mimicking the cell environment. KEY FINDINGS: Internal K(+) stimulated, while external Na(+) strongly inhibited the uptake of [(3)H]acetylcholine in proteoliposomes. Strong inhibition was exerted also by external K(+) while Mg(2+) or sucrose had no effect. Differently, the efflux of [(3)H]acetylcholine from proteoliposomes was not influenced by external or internal Na(+) and was only marginally stimulated by internal K(+). By dose response analysis of the Na(+) inhibition, an IC(50) of 1.3mM was derived. The kinetic analysis of the Na(+) effect revealed a competitive type of inhibition on acetylcholine uptake, i.e., Na(+) interacts with the same external binding site of acetylcholine with a Ki of 1.2mM. SIGNIFICANCE: Acetylcholine transport catalyzed by hOCTN1 revealed an asymmetric regulation by Na(+). Since the orientation of the transporter in the liposomal membrane is the same as in native membranes, and on the basis of sidedness of inhibition, physiological acetylcholine is principally exported by the transporter. This implies a role in autocrine and paracrine effects in non neuronal tissues.