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PURPOSE This study investigated the effect of toothbrush bristle stiffness and brushing force on the cleaning efficacy in vitro. MATERIALS AND METHODS Eighty bovine dentin samples were allocated to eight groups (n=10). Two custom-made toothbrushes of different bristle stiffness (soft and medium) were tested at four different brushing forces (1, 2, 3 and 4 N). Dentin samples were stained in black tea and brushed (60 strokes/min) for a total of 25 min in a brushing machine with an abrasive solution (RDA 67). Photographs were taken after 2 and 25 min of brushing time. Cleaning efficacy was measured planimetrically. RESULTS After 2 min of brushing, the soft-bristle toothbrush did not cause statistically significantly different cleaning efficacy at different brushing forces, while the medium-bristle toothbrush cleaned statistically significantly less efficaceously only at 1 N. Comparing the two different toothbrushes, higher cleaning efficacy was observed only at 1 N for the soft-bristle brush. At 25 min brushing time, the soft-bristle cleaned statistically significantly better at 4 N compared to 1 N and 2 N and at 3 N compared to 1 N. Using the medium-bristle, cleaning efficacy increased with increasing brushing force. After 25 min of brushing, no statistically significant difference was observed between the two different toothbrushes. CONCLUSION Irrespective the brushing force, the use of a soft or medium toothbrush results in comparable cleaning efficacy. At 2 min brushing time, increasing the brushing force does not increase the cleaning efficacy.