The aim of the present study is to investigate whether the chloride affects cell growth and cell-cycle progression of cancer cells. In human gastric cancer MKN28 cells, the Culture in the Cl-replaced medium (replacement of Cl- by NO3-) decreased the intracellular chloride concentration ([Cl-](i)) and inhibited cell growth. The inhibition of cell growth was due to cell-cycle arrest at the G(0)/G(1) phase caused by diminution of CDK2 and phosphorylated Rb. The culture of cells in the Cl-replaced medium significantly increased expressions of p21 mRNA and protein without any effects on p53. These observations indicate that chloride ions play important roles in cell-cycle progression by regulating the expression of p21 through a p53-independent pathway in human gastric cancer cells, leading to a novel, unique therapeutic strategy for gastric cancer treatment via control of [Cl-](i). (c) 2007 Elsevier Inc. All rights reserved.