Background: The antibacterial and anticollagenolytic properties of tetracycline (TCN) are valuable in periodontal therapy, and TCN treatment can remove the smear layer following root instrumentation. However, recent reports pointing to cytotoxic effects of several acids prompted this study to define TCN concentrations that are anticollagenolytic and remove the smear layer, but have low cytotoxicity. Methods: Human gingival (hGF) and periodontal ligament (hPDL) cells were treated short- (3 minutes) or long-term (24 hours) with TCN to determine concentrations yielding 50% (TD50) and 90% (TD10) cell survival. Activity assays measured TCN concentrations with half-maximal inhibition (IC50) of matrix metalloproteinase-2 and -9 (MMP-2 and -9). Finally, we analyzed the effects of TCN with high (75 mg/ml) or low (1 mg/ml) cytotoxicity on the smear layer by scanning electron microscopy (SEM). Results: The TD50 for TCN after short-term treatment was 4 mg/ml for both hGF and hPDL. Ninety percent of the cells survived 0.2 mg/ml. With long-term treatment, the TD50 for hGF and hPDL was 70 and 30 μg/ml, respectively, and the TD10 was 20 and 5 μg/ml. HGF and hPDL recovered from the 3-minute treatment with 1 mg/ml, but not from concentrations exceeding 3 and 9 mg/ml, respectively. The IC50 was 25 μg/ml for both MMP-2 and MMP-9. Whereas 75 mg/ml TCN removed the smear layer, 1 mg/ml TCN had no effects. Conclusions: Tetracycline has significant cytotoxicity on periodontal cells. Since non-cytotoxic concentrations of TCN inhibited MMP-2 and -9 but had no effects on the smear layer, TCN is not recommended for root surface treatment.