Inert fluorescent microparticles were used as tracers to investigate the dynamics of spatial distribution of particulate components in mixed population biofilms. The tracer bead spatial distributions in the biofilm were experimentally measured by sectioning the biofilms with a microslicer. The experimental results were compared with model simulations using the biofilm model (BIO-SIM) to evaluate the assumption that advective transport (displacement) of particulates balances with cell growth in the model. The tracer beads could traverse throughout a biofilm 360 ยตm thick within less than 23 minutes, which cannot be explained solely by their attachment to the surface followed by molecular diffusion. Advective transport of the tracer beads via ''voids and pores'' could be responsible for such rapid bead penetration. Observation by confocal scanning laser microscopy (CSLM) clearly showed that the biofilm consisted of a thick loose surface layer, varying in thickness, and a semicontiguous base layer separated by water channels. About 80% of attached tracer beads remained in the biofilm for over 20 days. The trapped tracer beads were gradually transferred from the depth of the biofilm to the surface. The observed bead release rate was much slower than the model predictions. This is probably because the cell density increased predominantly near the substratum, resulting in an unbalance of advective transport of the tracer beads and cell growth. The pores, voids, and cellfree spaces in the biofilm were first filled with growing biomass, thereafter, displacement of the beads took place once the cell density reached certain levels. The model assumptions of the temporal and spatial constant cell density and the continuum concept (flat biomass) are clearly oversimplified and should be revised. It was concluded that the dynamics of the inert microbeads in the biofilm was strongly influenced by not only microbial growth, but also by the biofilm structure and growth pattern. Therefore, one dimensional modeling is not adequate for the accurate description of the transport of particulates in a biofilm.