The aim of this study was to elucidate whether soft tissue reactions during chlorhexidine (CHX) treatment might be associated with meticulous oral hygiene measures. A commercially available 1% chlorhexidine digluconate gel (Hibitane Dental Gel) was applied in a custom tray for the mandibular arch, 10 min daily, for 14 days. A split mouth approach was used to allow evaluation of the effect of oral hygiene measures with toothbrush, toothpaste, and dental floss on one side of the arch. During the treatment period, 8 out of 12 subjects developed gingival and/or mucosal reactions appearing as white patches or ulcerations. Both the gingival reactions on the buccal marginal gingiva and the mucosal reactions near the margin of the custom tray occurred only when the gel treatment was combined with meticulous oral hygiene measures. The white patches, which could easily be detached, leaving ulcerated areas, consisted entirely of stratified squamous epithelium. The findings suggest that intensive treatment with chlorhexidine gel, in individually fitted custom trays, combined with meticulous oral hygiene measures may induce toxic effects on the surface layers of the gingiva and mucosa.