The production and modification of chitosan microparticles using crosslinking agents and papain were evaluated for controlled release applications. Chitosan microparticles were produced and crosslinked with sodium tripolyphosphate (TPP) 10% (w/v) solution or glutaraldehyde (GLU) 0.75% (w/w), with subsequent papain sorption. Microparticles were characterized by Fourier transformed infrared spectroscopy (FTIR) for chemical modifications, scanning electron microscopy (SEM) for morphology and X-ray diffraction (XRD) for crystallographic analysis. Chemical composition and the thermal stability of the material were characterized by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). It was observed that the presence of TPP and papain resulted in a decrease of the stability of the chitosan matrix. Papain release rates from the microparticles were also conducted in vitro. The amount of released papain in phosphate buffer (pH = 7.4) was analyzed with UV-spectroscopy, showing release profiles of enzymatic activity ranging from 0.006 to 0.011 mu mol.min(-1). The results indicate that both chitosan-TPP-papain and chitosan-GLU-papain microparticles can successfully be used for systems that aim at a controlled release of papain with potential use in the biomedical area. (C) 2010 Elsevier B.V. All rights reserved.