Background: The purpose of this in vitro study was to evaluate the effect of CO2 laser on the periodontally involved root surface, and to compare its efficacy with citric acid, EDTA and hydrogen peroxide in removal of root surface smear layer after root planing., Methods: The study was conducted on 50 periodontally involved single rooted human teeth with poor prognosis. Immediately after extraction the teeth were scaled and root planed with Gracey curets; 50 specimens were obtained from the proximal side of each tooth which were assigned randomly to 1 of the 4 groups. Group A (35 specimens) was divided into 7 subgroups of 5 specimens each and irradiated with CO2 laser using a defocused mode at 3-watt power for 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, or 1.4 seconds, respectively. Groups B, C, and D (5 specimens each) were treated with 6% hydrogen peroxide, EDTA solution (pH 7.4), or saturated citric acid solution (pH 1), respectively for 3 minutes. The specimens were then fixed and scanned using SEM., Results: It was observed that surface smear layer was present on root surfaces of teeth that were root planed; the CO2 laser was not able to remove the smear layer on the sites that were irradiated for 0.2, 0.4 or 0.6 seconds at 3W power. Irradiation time of 0.8 seconds at 3W power was able to remove the smear layer, but the dentinal tubules were partially exposed. The surface irradiated for 1 second showed a flat appearance with many clear orifice of dentinal tubules. No change in diameter of exposed dentinal tubules was observed and their diameter was nearly equal that of normal dentinal tubules. Irradiation time of 1.2 and 1.4 seconds produced surface charring and carbonization and was totally ineffective in exposing the dentinal tubules; 6% hydrogen peroxide did not remove the smear layer completely and the exposed dentinal tubules orifice was not clear. EDTA (pH 7.4) and citric acid (pH 1) were found to be effective in removing the smear layer and exposing the dentinal tubules, which showed funnel shaped widening., Conclusions: Surface smear layer was present on root surfaces of teeth that were root planed. Laser irradiation of 1 second at 3W completely removed the smear layer with minimal change in the diameter of the dentinal tubules. EDTA and citric acid were also effective in removing smear layer, but the exposed dentinal tubules showed funnel-shaped widening.