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Mild acids are known to activate dentin matrix metalloproteinase (MMPs). All self-etching dental adhesives are acidic (pH 1.5–2.7) and may activate dentin MMPs. The purpose of this study was to compare the ability of several all-in-one adhesives to activate gelatinolytic and collagenolytic activities in powdered mineralized dentin. Powdered dentin made from human teeth was mixed with all-in-one adhesives (Clearfil Tri-S Bond, G-Bond, Adper Prompt L-Pop) or a self-etching primer (Clearfil SE Bond primer) for varying times and then the reaction was stopped by extracting the adhesives using acetone. Fresh untreated mineralized dentin powder had a gelatinolytic activity of 3.31 ± 0.39 relative fluorescent units (RFU) per mg dry weight (24 h) that increased, over storage time, to 87.5 RFU mg−1 (24 h) after 6–8 wk. When fresh powder was treated with acidic Tri-S Bond, the gelatinolytic activity increased from 3.24 ± 0.70 RFU mg−1 to > 112.5 RFU mg−1 (24 h) after 20 min and then remained unchanged. Monomers with lower pH values produced less activity. There was a significant, direct correlation between gelatinolytic activity and pH, with Tri-S giving the highest activity. Coating dentin powder with Tri-S resin prevented fluorescent substrates from gaining access to the enzyme, even though it activated the enzyme. In conclusion, self-etch adhesives may activate latent MMP and increase the activity to near-maximum levels and contribute to the degradation of resin–dentin bonds over time.