The nasal cavity has received much attention as a an alternative route for systemic administration of drugs with poor bioavailability and biosensitive and high molecular weight compounds such as proteins, peptides, steroids and vaccines (Arora et al., 2002), and as a possible route for direct brain-targeting of drugs (Illum, 2004). The use of chitosan is an interesting approach in nasal drug delivery since it is a mucoadhesive polymer considered biodegradable and biocompatible with low toxicity (Sinha et al., 2004). The aim of this study was to design, prepare and evaluate rokitamycin-loaded microspheres containing different types of chitosan for nasal administration and brain-targeting of the drug.Sixteen microparticulate formulations containing chitosan base, chitosan glutamate, or two chitosan derivatives, N+-chitosan-7 and N+-chitosan-8, loaded with rokitamycin were prepared using a spray-drying technique. Different preparation parameters were set; the polymer to drug ratios 4:1 and 5:1 and the feed solution concentrations 0.25% and 1.0%. The microspheres were characterised with respect to properties such as drug content, particle size and particle size distribution, morphology and in vitro release of rokitamycin. The water uptake was investigated on four selected formulations and the corresponding drug-free formulations. It was further carried out a mucoadhesion test on the four selected formulations. The effect of the different chitosans and the different preparation parameters on the microspheres properties was explored. Finally, an in vivo experiment was performed to evaluate chitosan microspheres as a nasal drug delivery system for brain-targeting of rokitamycin.This study shows that rokitamycin-loaded chitosan microspheres can be prepared by a spray-drying technique and that the type of chitosan, concentration of feed solution and polymer to drug ratio influence the characteristics of the microspheres. The formulations have properties suitable for nasal administration and are promising as delivery devices for direct nose-to brain targeting of rokitamycin. Drug-loaded chitosan glutamate microspheres prepared from a 1% feed solution with the polymer to drug ratio 4:1 show promising results in vivo; rokitamycin is absorbed into the bloodstream after nasal administration. Further, preliminary results show that rokitamycin is able to reach the central nervous system after nasal administration of loaded chitosan glutamate microspheres.