Effect of calcium ions (Ca++) on the virulence of Shigellaflexneri 2a was examined with reference to its infectivity to cultured cells, the guinea pig eye, and the ligated small intestine of rabbits. The organism grown in a calcium-containing medium showed a significantly higher ability to penetrate HeLa cells than that of organism grown in a calcium-deficient medium. This ability was constantly maintained in the presence of Ca++, while readily lost in the absence of Ca++. Similarly, its pathogenicity for the guinea pig eye and the ligated small intestine of rabbits was more or less greater in cases with Ca++ than in cases without Ca++. Ca++ seemed to play an important role in bacterial adhesion to the cell membrane, by which the cellular engulfment was induced. The presence of Ca++ in the environment of bacterial growth was emphasized on development and maintenance of virulence of S. flexneri 2a.