Purpose: The use of Er:YAG lasers for intracanal ablation of dentine, as part of biomechanical preparation in endodontics, is attracting interest. However, this process is yet to be optimized fully in terms of mode of operation, delivery system, and irrigation. Water irrigation is useful for reducing thermal stress to dental hard and soft tissues during laser treatment. The aim of this study was to compare the ablation characteristics of dentine when ablated from the root canal (radicular) aspect compared with the external root (periodontal) aspect, and examine the effect of water mist spray flow rate on the efficiency of ablation. The application of these parameters to direct ablation of smear layer in the root canal environment was then tested, comparing lasing with a 400-孠fiber to the passive effects of EDTA during and after rotary Ni-Ti preparation. Materials and Methods: In part 1, single-rooted extracted teeth were split into two parts, and the surfaces of the split roots irradiated at 1 Hz for 5 pulses with a KaVo KEY3 Er:YAG laser at pulse energies of 250, 300, 400 or 500 mJ. This was undertaken with either a low (0.5 ml/min) or high (1.5 ml/min) water flow rate. A total of 10 sites were irradiated for each of the 16 unique energy/water flow rate/site combinations, giving 160 sites. Crater depth, diameter, and volume were measured, and differences between groups also assessed using light microscopy and SEM. In part 2, root canals prepared using rotary Ni-Ti instruments were irradiated in the presence of water using an optical fiber, and the effects on dentine assessed using image analysis. Results: All dentine sites showed ablation at 250 mJ/pulse. There was a consistent increase in crater depth and diameter with increasing energy in all subgroups, with larger craters with low water flow than with high water flow on the external (periodontal aspects). Comparing the effect of location, there was significantly greater ablation on the periodontal aspect in the low water flow group than on the root canal (radicular) aspect. In contrast, in the high water flow group, there was no significant difference between the two locations. Irradiated surfaces had open dentinal tubules but no carbonization, cracking or other microscopic types of surface thermal injury. Lasing in the canal with an optical fiber in the presence of water had a limited and irregular effect on a thick smear layer, when compared to the passive effect of EDTA. Conclusion: These results indicate that significant interactions occur between dentine ablation and the variables of water spray flow rate and dentine location. This will be useful for developing methods to ensure that sufficient energy can be delivered to radicular dentine to achieve effective ablation for the biomechanical preparation of root canal dentine. Excess water can attenuate effective dentine ablation with the Er:YAG when used in the root canal.