The dynamics of spatial distribution of particulate components in mixed population biofilms was investigated using lμm fluorescent microparticles as tracer. Shifts of the tracer bead spatial distribution in the biofilm were measured by sectioning the biofilm with a microslicer and were compared with model simulations to evaluate the growthadvection concept in the existing biofilm models. The tracer beads could traverse throughout a 360 μm thick biofilm within 23 minutes, could be attributed to advective transport via water channels and pores. The release of the entrapped beads were much slower than predicted by a one dimensional model due to spatial and temporal changes in cell density in the biofilm. This evidence implied that cell growth and advection was not balanced due to the presence of biofilm heterogeneity (e. g., pores and voids). Three dimensional observation by a confocal scanning laser microscope clearly indicated that the biofilm consisted of semicountiguous base film and relatively high thickness variation of loose surface film. The substratum was not uniformly covered by the biofilm and cell-free spaces and voids were observed near the substratum. This suggested that the pores, voids and cell-free spaces in the biofilm were firstly filled with growing biomass, thereafter displacement of the tracer beads took place once the cell density reached certain levels. Model assumptions of constant cell density and a continuum concept (flat biomass) are clearly over simplified and should be revised. It was concluded that microbial population dynamics in the biofilm can be determined by not only microbial growth kinetics and physiology, but also by the biofilm structure and growth pattern. One dimensional approach (modeling) is, therefore, adequate to predict biofilm accumulation and its performance, but inadequate to accurately describe the microbial population dynamics in the biofilm.