A fluidized bed denitrifying reactor was run to examine the vertical segregation of sand particles on the basis of different biofilm coverage, so far neglected when modelling fluidized beds, The segregation was found to be significant and it can be directly correlated with the vertical hydrostatic pressure profile in the bed. A procedure was developed for the rapid determination of biofilm thickness from hydrostatic pressure data using a recently published method based on the use of the novel criteria "expansion coefficient" and "specific occupied volume". A key feature of the procedure is the "particle content", which can be calculated from particle characteristics and is correlated in this study with the hydrostatic pressure gradient. The method was verified by directly measuring biofilm thickness as a function of the vertical position in the bed. This way biofilm thickness can be calculated from a readily measurable hydrostatic pressure profile with an error of 0.04-0.06 mm. This error is believed to be due to N-2 gas entrapment in the denitrifying biofilm and to the original inaccuracy of the determination of particle size and volume. The method is rather insensitive to the exact biofilm density when the usual high-density carrier material is used.