BACKGROUND: Gingival bleeding is frequently accompanied by an ulcerated epithelium, with repair depending on the proliferative activity of the epithelial cells. The present study examined the proliferative activity of the junctional epithelium (JE) under several different methods of toothbrushing stimulation. METHODS: Twelve dogs were used in this 3-week experiment. Prior to the experiment, all teeth underwent daily removal of plaque and calculus using a scaler. Teeth were then divided into quadrants: 9 teeth in 3 quadrants (test, 3 per quadrant) were mechanically stimulated by toothbrushing for various time intervals (10, 20, or 40 seconds) and at various forces (100, 200, or 250 gf). The 3 teeth in the fourth quadrant served as controls. The proliferative activity of the basal cells of the junctional epithelium was evaluated for expression of proliferating cell nuclear antigen (PCNA). The numbers of PCNA-positive basal cells and total basal cells were counted, and the width of the junctional epithelium was measured. RESULTS: Toothbrushing force significantly affected the PCNA-positive basal cell ratio (P < 0.05). The 200 g toothbrushing force produced the highest PCNA-positive basal cell ratio, which was significantly higher than that using the 100 g force (P < 0.05). CONCLUSION: In junctional epithelium stimulated with a toothbrush, the PCNA-positive basal cell ratio is more sensitive to toothbrushing force than to duration.