Purpose: Radiation-related caries is characterized by enamel delamination near the dentinoenamel junction (DEJ). We investigated the activity and expression of the matrix metalloproteinases (MMPs) -2 and -9 in order to understand disease pathogenesis in teeth submitted or not to radiotherapy (RT).<br/> Methods: In situ zymography and immunofluorescence assays were performed to evaluate the activity and expression of MMPs -2 and -9, respectively. Twelve primary second molars were randomly assigned into two experimental subgroups: irradiated and nonirradiated. Dental fragments were exposed to radiation at a dose fraction of two Gy for five consecutive days until reaching the total dose of 60 Gy. The percentage of fluorescence in the DEJ was evaluated in three distinct regions of the tooth (cervical, cusp, and pit). The regions were photographed under fluorescence microscopy at 1.25× and 5× magnification.<br/> Results: The intensity of fluorescence per mm 2 in the DEJ was higher in the cervical region of irradiated primary teeth (P <0.05) versus nonirradiated ones. In these areas, immunofluorescence revealed expression of MMPs -2 and -9.<br/> Conclusion: Radiotherapy can increase the activity of MMPs -2 and -9 in the cervical region of the DEJ of primary teeth.