Root conditioning has introduced to dissolve the smear layer and to produce surface demineralization, resulting to exposure of dentin collagen fibril and opening of dentinal tubules. The purpose of the present study was to examine the effect of different concentration and application time of tetracycline-HCL on root conditioning. Total 40 root specimen were prepared from 20 periodontitis-prone human single rooted tooth. The specimen were treated with tetracycline-HCL solution(20mg/ml, 50mg/ml, 100mg/ml)for 20 sec, 3 min, 5 min., and saline for 30 sec. The application method was rubbing method with cotton pellet. Under the scanning electron microscopy(20KV), the extent of smear removal and opening of the dentinal tubules were examined at x 3000. The following results were obtained. 1. Treatment of root specimen with saline did not remove the smear layer and open the dentinal tubules. 2. Treatment of root specimen with different concentration of tetracycline-HCL for 20 sec also did not remove the smear layer completely. 3. Treatment of root specimen with different concentration of tetracycline-HCL for 3 min opened the dentinal tubules and removed smear layer. 4. Treatment of root specimen with 50mg/ml of tetracycline-HCL for 3 min showed collagen fibril within the opened dentinal tubules. In conclusion, the effect of root conditioning with tetracycline-HCL is more dependent on the application time than the application concentration. Root conditioning with 50mg/ml tetracycline-HCL for 3 min is enough for obtaining the periodontal regeneration.