Hydrogen sulfide (H₂S) is not only one of the main causes of halitosis but is also an agent of toxicity against periodontal cells and tissues in biofilm-related periodontal diseases. Also, apoptosis of gingival epithelial cells may play an important role in the onset and progress of periodontitis. We examined the effect of H₂S on the induction of apoptosis, using human gingival fibroblasts (HGF) and keratinocyte-like Ca9-22 cells derived from human gingiva. The cells were incubated with H₂S (100 ng ml⁻¹) for 24, 48 or 72 h by adding H₂S to air containing 5% CO₂, supplied constantly to the culture environment during incubation. The incidence of apoptosis caused by H₂S was determined with Annexin V staining by flow cytometry. The proportion of apoptotic cells was significantly increased by exposure to H₂S for 48 h in comparison with the control in both Ca9-22 cells and HGF. A concentration of 100 ng ml⁻¹ H₂S in air is possible in the gingival sulcus. This study indicates that apoptosis in gingival epithelial cells and HGF by H₂S may occur in the oral cavity, which may cause a periodontal condition.