The purpose of this study was to investigate the efficiency of hand instrumentation and laser irradiation on calculus removal from the root surfaces, in vitro. Thirty-two human teeth, extracted for periodontal reasons, were used in this study. Root surfaces of single-rooted teeth were treated by different methods including (1) conventional hand instruments; (2) hand instruments and tetracycline-hydrochloride (Tet-HCl); (3) erbium, chromium:yttrium-scandium-gallium-garnet (Er,Cr:YSGG) laser irradiation, setting I (short pulse); (4) Er,Cr:YSGG laser irradiation, setting II (long pulse). Three premolar teeth, extracted for orthodontic reasons, served as control. Themorphology of the root surfaces was evaluated by light and scanning electron microscopy. Energy dispersive X-ray (EDX) analysis was performed to compare the mineral content of root surfaces treated with hand instrumentation and lasing procedures. The results of this study demonstrated that all treatments were efficient in calculus removal from the root surfaces. Thermal changes, including melting and carbonization, were not observed in either lasing procedure. The surface was rougher in the laser groups than in the groups treated with hand instruments. Moreover, roughness was greater in the long-pulse laser setting than in the short-pulse setting. While increased calcium (Ca) and decreased phosphate (P) (weight concentration percent) were observed in all treatments when compared with the control, laser procedures resulted in a more similar mineral content than in the groups treated with hand instruments. Based on these findings, laser procedures, when used in appropriate settings, are capable of performing scaling and root planing in the treatment of periodontitis. It may be concluded that short pulse laser may be more suitable for the micro-morphology of the root surface. However, additional in vitro and clinical studies are necessary to clarify the success of laser in periodontal therapy. © Springer-Verlag London Limited 2009.