IN THIS STUDY, SUBSTANTIVITY OF topically applied doxycycline hydrochloride on root surfaces obtained from patients with periodontal disease was studied in vitro. Cementum and dentin specimens were impregnated with aqueous solutions of doxycycline for 3 minutes, and incubated in serum for 10 minutes, 7 days, and 14 days. Substantivity was determined by agar diffusion inhibition assay. Comparative analyses of cementum versus dentin groups were conducted with 2-way analysis of variance. Results indicated that there were no statistically significant differences between the binding potential of doxycycline to cementum and dentin specimens. Antibacterial effect of specimens treated with the concentration of 100 mg/ml persisted for 14 days, indicating the presence of doxycyline in biologically active form. Our findings demonstrate the long lasting substantivity of doxycycline hydrochloride on periodontally diseased root surfaces and supports the concept of using root surfaces as a substrate for the deposition and slow release for local tetracycline delivery.