The Er:YAG laser may be used on periodontally involved teeth in combination with conventional periodontal therapy in order to improve the efficacy of root instrumentation. The aim of this study was to compare the effect of hand instrumentation on root surfaces of periodontally involved teeth with Er:YAG laser application. Thirty freshly extracted, non-carious, single-rooted, periodontally diseased human teeth from adult humans with advanced periodontal disease were used in this study. The teeth were divided into three groups of 10 specimens each. Group A was treated with scaling and root planing (SRP) with curets only (control). In group B, the root surfaces were scaled with curets and then lased with an Er:YAG laser (wavelength 2.94 microm). A handpiece with a water spray was used in non-contact mode (defocused) at a distance of 1 cm from root surface. Laser parameters were set at energy of 100 to 200 mJ/pulse, with 10 Hz of frequency. In group C, the root surfaces were lased only with power settings 250 to 300 mJ/pulse and 10 Hz frequency. An epon-araldite plastic embedding technique was used for light microscopic investigation. Histologic findings showed significant differences between the test and control sites. In control sites, after hand instrumentation, the surface was smooth, without a cementum layer, and the dentin layer presented opened tubules. Defects on the dentin layer were also present along root surfaces. In the test sites (B, C) root surfaces revealed no thermal damage; no cracking or tissue carbonization were observed. The superficial layers of lased surfaces appeared smooth and melted without alterations. Based on these findings, it appears that it may be feasible to use the Er:YAG laser for root instrumentation without prior root planing if the proper parameters are followed.