This study investigated the penetrability of dentinal tubules in cavity walls lined with different dentin bonding systems. Occlusal Class I cavities were prepared in 93 premolars. The cavities in the control group had an intact smear layer without a lining, while those in the experimental group were lined with Gluma CPS, Scotchbond Multi-Purpose Plus or One-Step. The penetrability of the dentinal tubules was tested with a dye (basic fuchsin) or bacteria (S faecalis) immediately after adhesive lining and after one-month storage in water at 37 degrees C. Some of the lined samples were sectioned and examined under the SEM. In some samples in the experimental group, the dye penetrated to the pulp and bacteria up to 125 microm into the dentinal tubules immediately after lining. The Kruskal Wallis ANOVA and Tukey test showed the depth of dye and bacterial penetration to be significantly less in teeth with bonding systems than those in the control group (p<0.05). However, there was no statistically significant difference between the control and experimental groups after storage in water (p>0.05). SEM examination showed that the hybrid layer and resin tags were present in the cavity walls immediately after lining but absent after storage in water. Therefore, adhesive linings under the experimental conditions were ineffective in preventing dye or bacterial penetration of the dentinal tubules.