The aims of the present study were: a) to assess the impact of the intraoral location on the rate of biofilm growth, and b) to establish an in vivo biofilm model to examine intraoral biofilm growth. Eight healthy volunteers wore acrylic splints with 15 glass slabs each in the upper and lower jaws to build up plaque. After 48 h, the specimens were removed and stained using the vital fluorescence technique. Biofilm thickness was evaluated by confocal laser scanning microscopy (CLSM). The mean plaque thickness amounted to 77.6 +/- 29.1 microm on the buccal sites of the upper jaw and 71.9 +/- 26.3 microm on the buccal sites of lower jaw. On the palatal site a biofilm of 52.1 +/- 26.2 microm thickness was grown, which was significantly less compared with the other locations evaluated (p < 0.001). The results demonstrate that the in situ biofilm thickness on the buccal sites was similar irrespective of the location in the oral cavity. The new splint system described may be a useful tool for further standardised experimental studies regarding influences on growth and structure of intraoral biofilms.