Background: A series of animal and in vitro data confirms that nicotine impairs bone healing, diminishes osteoblast function, and causes autogenous bone graft morbidity. Therefore, this study aimed to investigate the impact of nicotine on the healing of bone defects treated by the guided bone regeneration (GBR) principle. Methods: Sixteen mongrel dogs were used. One defect was surgically created bilaterally and randomly assigned as an expanded polytetrafluoroethylene (ePTFE) membrane site or a non-membrane control site. The animals were randomly assigned to one of the following groups: group 1, placebo (n = 8) and group 2, subcutaneous administration of nicotine (2 mg/kg) twice daily (n = 8). After 4 months, the animals were sacrificed and the specimens routinely processed for semi-serial decalcified sections. The evaluated parameters were bone height, bone width, bone density, and bone area of newly formed bone. Results: Intergroup analysis (Kruskal-Wallis) showed that membrane-protected defects in the placebo group demonstrated an increased bone area when compared to membrane-protected defects in the nicotine group and non-membrane sites, regardless of nicotine administration (P<0.05). In addition, nicotine administration significantly affected bone density in membrane- and non-membrane-protected sites (P<0.05). Conclusions: Within the limits of the present study, nicotine might affect, but not prevent, bone healing in defects treated by guided bone regeneration. The mechanisms of this effect should be investigated further.754565571