INTRODUCTION: The capacity of dentin and collagen to bind DNA and protect against spontaneous and nuclease-induced degradation was evaluated individually and by the incubation of DNA with nuclease-producing bacteria in a mixed culture. METHODS: Extracted Fusobacterium nucleatum DNA was incubated with dentin shavings or collagen for 90 minutes. The DNA-bound substrates were incubated in different media (water, sera, and DNase I) for up to 3 months. Amplifiable DNA was released from dentin using EDTA,or from collagen using proteinase K, and evaluated by polymerase chain reaction (PCR). The stability of dentin-bound DNA was also assessed in a mixed culture (Parvimonas micra and Pseudoramibacter alactolyticus) containing a DNase-producing species, Prevotella intermedia. Samples were analyzed for amplifiable DNA. RESULTS: In water, dentin-bound DNA was recoverable by PCR at 3 months compared with no detectable DNA after 4 weeks in controls (no dentin). DNA bound to collagen was detectable by PCR after 3 months of incubation in water. In 10 human sera, amplifiable DNA was detectable at 3 months when dentin bound and in controls (no dentin). In mixed bacterial culture, dentin-bound DNA was recoverable throughout the experimental period (3 months), compared with no recoverable F. nucleatum DNA within 24 hours in controls (no dentin). CONCLUSIONS: There is a strong binding affinity between DNA and dentin, and between DNA and serum proteins or collagen. These substrates preserve DNA against natural decomposition and protect DNA from nuclease activity, factors that may confound molecular analysis of the endodontic microbiota yet favor paleomicrobiological studies of ancient DNA.