Extracellullar cysteine-proteinases, referred to as gingipains, are important virulence factors for Porphyromonas gingivalis, an anaerobic gram-negative bacterium recognized as a major etiologic agent of chronic periodontitis. Antibiotics have been used in dentistry as adjuncts to periodontal therapy. Tetracycline and its analogues as protein synthesis inhibitors in prokaryotes, are important antibiotic agents against a broad spectrum of anaerobic gram-negative bacteria. Our aim in this study was to measure enzyme activity of gingival exudates on the fluorogenic substrate Pac-Phe-Arg-MCA and investigated the inhibitory effect of antibiotics tetracycline and its analogues doxyclycline, minocycline and oxytetracycline on the enzymatic activities. The proteolytic activity of gingival exudates enzymes was determined in control individuals (n=15) and patients with clinical periodontitis (n=30). The samples was incubated in 0.2 M Tris-HCl buffer solution (pH 7.5) with 50 mM Gly-Gly, 10 mM L-Cys, 5 mM CaCl₂ and 21.5 uM Pac-Phe-Arg-MCA followed by pooled samples according to enzymatic activity levels. The substrate hydrolysis was measured by following fluorometrically (l_ex=380nm, l_em=460nm) the time-course appearance of MCA in solution. To investigate the inhibitory effect of tetracycline and its analogues on enzyme activity, pool of gingival exudates of patients with periodontitis, were incubated with various concentrations (0.01 uM – 100 uM) of tetracycline and its analogues as well as the concentration of these compounds required to inhibit 50% of the enzyme activitivy (IC₅₀) was measured. Thus, the IC₅₀ for tetracycline, doxyclycline, minocycline and oxytetracycline were 9 uM, 30 uM, 53 uM and 85 uM, respectively. These results indicate tetracyclines as cysteine-proteinases inhibitors, including gingipains, and may explain the therapeutic efficiency of these antibiotics in the treatment of periodontitis.