Endogenous dentinal matrix metalloproteinases (MMPs) have been implicated in the auto-degradation of collagen fibrils within resin infiltrated layers of dentinal attachment. In order to target these proteinases, we must know which MMPs are produced and activated at the resin/dentin interface. In this study, we have optimized an extraction procedure and quantitated levels of endogenous MMPs in samples of dentin removed from the cavity walls of a single, extracted tooth. In our tooth-cavity model, an occlusal cavity (2×4×2 mm) was prepared and removed from the tooth crown, leaving surrounding dentinal walls of 1-mm-thick. The samples were pulverized with an analytic mill. Using enzyme-linked immunosorbent assay (ELISA), an average of 34.7 picograms of MMP-9 was detected in less than 300 mg of dentinal powder. This is the first study of its kind to quantitate endogenous levels of MMP in dentinal protein isolated from the cavity walls of a single, extracted tooth.