The goal of this research is to develop the technology platform required for the production of bioactive paper based on enzymes as bioactive agents. The immobilization platform described here is based on microencapsulation, which consists in the entrapment of biomolecules in the core of hollow spheres made by a semipermeable membrane. The capsules containing the enzymes can be either deposited on paper or mixed with paper pulp to prepare a bioactive paper. The activity of encapsulated laccase was compared with that of free enzyme using its reaction with the o-phenylenediamine (OPD) substrate. Confocal Laser Scanning Microscopy (CLSM) is used to study the location of protein in microcapsules and provides explanations for differences in activity of encapsulated laccase. The location of protein in microcapsules was determined using BSA modified with the fluorescent tag sulforhodamine. Polyethyleneimine microcapsules were modified with fluorescein isothiocyanate allowing the simultaneous identification of capsule walls and of encapsulated proteins. From CLSM analysis, proteins were found to favor the wall of the capsules because of strong ionic attraction with the charged polymer. BSA was found to some extent in the core of the capsules and encapsulation of higher loadings increased the proportion of core proteins. We will also present our results on the incorporation of microcapsules in a paper substrate. CLSM was used in this section to determine the distribution and density of tagged microcapsules in the paper substrate. The response of immobilized laccase to a common substrate will also be described. (C) 2008 Wiley Periodicals, Inc. J Appl Polym Sci 111: 1-10, 2009