PURPOSES This study evaluated the effect of gamma radiation on the microtensile bond strength of resin-based composite restoration to human enamel and dentin performed either before or after radiotherapy. METHODS Thirty sound human third molars were sectioned mesio-distally into buccal and lingual halves and then randomly divided into enamel or dentin groups. Enamel and dentin substrates were randomly divided into three sub-groups (n = 10) Control, which received no irradiation; specimens were irradiated before restoration protocol; and specimens were irradiated after restoration protocol. Radiation therapy was defined by application of 60-Gy dose fractionally with daily exposures of 2 Gy, 5 days a week, over 6 weeks. Restorations were carried out using Adper Single Bond adhesive system and Filtek Z250 resin composite. The specimens were sectioned producing 4 sticks per specimen and submitted to microtensile on a testing machine. Data were submitted to two-way ANOVA followed by Tukey test (p RESULTS Bond strength to enamel was significantly higher than to dentin irrespective radiation therapy. Radiotherapy applied before restoration significantly reduced the bond strength to both substrates. A predominance of adhesive failures was detected for control groups and groups restored before radiotherapy. Cohesive failures in dentin and enamel increased when the specimens were restored after irradiation. CONCLUSIONS The gamma radiation had a significant detrimental effect on bond strength to human enamel and dentin when the adhesive restorative procedure was carried out after radiotherapy.