The mechanism of tetracycline-induced inhibition of matrix metalloproteinases (MMP) was studied by measuring the MMP secretion and MMP-2 mRNA levels in unkeratinizing periodontal ligament epithelial cells and skin keratinocytes cultured in the presence of doxycycline or chemically modified tetracyclines (CMT) lacking antimicrobial activity. Doxycycline, CMT-1, and CMT-8 exerted a direct dose-dependent inhibition of porcine periodontal ligament epithelial cell medium MMP activity as assayed by gelatin enzymography. Both the 92-kDa (MMP-9) and 72-kDa (MMP-2) gelatinases were inhibited by the tetracyclines added to the conditioned medium. Culturing the cells in the presence of the tetracyclines required considerably smaller concentrations to reduce the secreted MMP activity. The drugs were not toxic to the epithelial cells at concentrations from 4 to 250 micrograms/mL up to 24 h of culture. Tetracycline effects on the MMP-2 mRNA levels were studied in human skin keratinocytes using Northern hybridization analysis with a specific cDNA probe. A marked inhibition in the MMP-2 gene expression was observed by 6 h with 5 micrograms/mL of doxycycline, CMT-1 or CMT-8. Doxycycline inhibition was somewhat stronger than the two other tetracyclines. After 24 h of culture with 50 micrograms/mL of the drugs, the total RNA levels also decreased by 33 to 40%. The 72-kDa gelatinase activity in culture medium of the keratinocytes followed roughly the pattern of inhibition of the gene expression. We conclude that doxycycline and the chemically modified tetracyclines, in addition to inhibiting the MMP activity may also reduce the enzyme expression at the transcriptional level.