The purpose of this investigation was to evaluate the effects of chlorhexidine (CHX) on the attachment and growth of human gingival fibroblasts and periodontal ligament cells using an in vitro system where periodontal cells were grown on root surfaces treated previously with CHX. Our results indicate that the attachment of periodontal cells onto root surfaces was not adversely affected when roots were treated for 15 minutes with up to 0.12% CHX. However, cell attachment and morphology were adversely altered with prior 0.2 to 2.0% CHX treatment. The cells appeared round and retracted from roots treated with 0.2% CHX. With 2% CHX treatment, the cells exhibited a foamy appearance in which most of the cytoplasm seemed to have been extracted from the cells. Although 0.12% CHX treatment did not adversely affect the attachment of periodontal cells onto roots, direct exposure to as little as 0.01% CHX caused a 90% reduction in 3H-thymidine incorporation by cultured gingival fibroblasts. We conclude that although 0.12% CHX did not inhibit the attachment of cultured periodontal cells to pretreated roots, direct exposure of cells to much lower concentrations of CHX (0.0025 to 0.01%) caused a dose-dependent inhibition of growth.