The purpose of this study was to investigate the penetration of smeared and nonsmeared dentine by Streptococcus gordonii. Prepared human roots, grouped as either nonsmeared or smeared, were immersed in a suspension of S. gordonii cells for 3 weeks. The roots were then prepared for scanning electron microscopy and histological analysis. Dentine discs prepared from coronal dentine were grouped similarly. Using a fluid filtration apparatus, the hydraulic conductance (Lp) of each disc was determined before and after incubation with bacterial suspension. Scanning electron microscopy evaluation of the roots following infection with bacteria showed no change in the smear layer (P<0.0001). Histological sections revealed that bacterial penetration of all the nonsmeared samples had occurred, while nine out of 10 smeared samples showed no bacterial penetration (P<0.0001). The Lp of the nonsmeared discs was significantly reduced by 42% (P<0.0001) after bacterial penetration. However, the smeared samples revealed a 1% reduction in Lp which was not significant (P > 0.05). The results suggest that dentinal smear layers are an effective barrier to dentinal tubule invasion by S. gordonii.