The aim of this study was to determine the effects of varying parameters of Er:YAG laser irradiation with and without water spray cooling on root canal dentine in vitro. After horizontally removing tooth crowns from extracted human teeth, roots were axially sectioned into thin slices, exposing the root canal surface. An Er:YAG laser delivered 10-30 J/cm(2) into a 0.4-mm diameter laser spot on the root canal surface. Single pulses of different lengths (80-280 micro s) were applied with and without water spray cooling/irrigation, and sequences of three pulses at a repetition rate of 30 Hz were applied at selected pulse parameters. The irradiated samples were investigated using both confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). At most irradiation conditions, the root canal dentine surface was ablated. Three-dimensional images from CLSM revealed that the cavity walls were not smooth. Depths of the cavities revealed significant differences between the cavities. No debris was observed at the surface of cavities at any irradiation condition. Strong melting and recrystallisation, or unusually flat surfaces with open dentinal tubules were obtained with sequences of three pulses without water cooling. CLSM is an effective tool for investigation of laser effects on root canal dentine. By varying the irradiation conditions, the Er:YAG laser can induce different modifications of root canal surface, which may be very interesting for root canal preparation.