The role of proteolytic enzymes in the root caries process remains unclear. The aim of this study was to investigate collagenase activity during tooth root demineralization and remineralization in an in vitro demineralization/remineralization pH-cycling model, Human tooth roots were subjected to pH cycling (alternating demineralization and remineralization) in one of two different time cycles for five days. Collagenase at 90, 180, or 360 micrograms per root was placed into either the demineralizing solution or the remineralizing solution in the pH-cycling system. The effects of additional exposure to collagenase before or after pH cycling were also studied. After the exposure, thin sections of the roots were examined histologically by polarized light microscopy. Changes of calcium and phosphate in the solutions were analyzed chemically. Surface erosion occurred only in the groups where collagenase was contained in the remineralizing solution and in which the root samples were exposed to severe demineralization. However, no differences among the control and experimental groups were found in calcium and phosphate changes in the pH-cycling solutions. These findings suggest that collagenase works during the remineralizing phase and predominantly attacks the organic matrix of the root after demineralization. Additional exposure to collagenase before or after pH cycling did not increase surface erosion except for exposure to collagenase in the absence of phosphate following pH cycling.