The loosening of dental implants is associated with peri-implant vertical bone loss. The mechanisms and mediators of this bone destruction are not known. To test the hypothesis that collagenase-2 and collagenase-3 might be markers or maybe even mediators in this process, we measured collagenase-2 (time-resolved immunofluorometric assay) and collagenase-3 (quantitative immunoblot) in peri-implant sulcus fluid in 49 implant sites in 13 patients. Vertical bone loss was graded as being < 1 mm, from 1 to 3 mm, or > 3 mm. The severity of inflammation, as rated according to Gingival Index, did not correlate with the category of bone loss (p > 0.05). Collagenase-2 and collagenase-3 were higher (p < 0.05) in the group which had lost > 3 mm of bone than in the two other groups. Gingival Index is not a clinically important marker for bone loss, but collagenase-2 and collagenase-3 in peri-implant sulcus fluid are. They might participate in peri-implant osteolysis.