The antimicrobial action of acriflavine, a quaternary ammonium compound, on Staphylococcus aureus was studied by electron microscopic observation. The bactericidal activity of acriflavine was dose-dependent over the 4 hr of exposure time. Scanning electron micrographs showed a wavy wrinkled cell surface following treatment with acriflavine. Transmission electron micrographs showed thickened cell walls following treatment with acriflavine. Acriflavine-induced cell wall thickness seemed to affect both the peripheral and cross walls, but was reversible after treatment removal. These findings indicate that cell wall thickness is a characteristic phenotype of S. aureus exposed to acriflavine. It is therefore believed that cell wall thickness plays an important role in the mechanism of action of acriflavine.