A novel method of detecting extracellular protease activity at biofilm-substratum interfaces was developed. This method utilizes fluorescent molecules bound to cellulose substrata with a lectin. Extracellular proteases degrade the lectin and release the fluorochrome into solution. This new technique and a standard dissolved-substrate assay detected similar responses of biofilm extracellular protease activity to experimental manipulation of N supply. Combination of this technique with confocal scanning laser microscopy allowed direct visualization of microspatial patterns of bacterial distribution and extracellular protease activity at the biofilm-substratum interface.