Chitosan microspheres containing 5-fluorouracil (5-FU), tegafur (FT), and doxifluridine (DFUR) were prepared by the dry-in-oil method using silicone oil with no surfactant as a dispersion medium. For DFUR-containing chitosan microspheres (DFUR-M), reacetylation with acetic anhydride or coating using chitosan and glutaraldehyde was performed. DFUR-M, reacetylated DFUR-M, and chitosan-coated DFUR-M were investigated on in vitro drug release, and the former two microspheres were examined for in vivo degradation after subcutaneous (s.c.) implantation in mice, and in vivo plasma concentration-time profiles after s.c. implantation in rats. The present method gave fairly large microspheres purely composed of chitosan and drug because of no use of surfactant, which showed the mean particle diameters of 300-900 microm and the drug contents of 4-22% (w/w). Encapsulation efficiency of DFUR was higher than that of 5-FU and FT. DFUR-M and reacetylated DFUR-M exhibited spherical shape except chitosan-coated DFUR-M. DFUR-M showed high initial rapid release, which was suppressed to some extent by reacetylation or chitosan coating. DFUR-M and reacetylated DFUR-M subcutaneously implanted were gradually degraded, and approximately half or a little more of the microspheres disappeared from the implanted site at 3 weeks postimplantation. DFUR-M and reacetylated DFUR-M implanted subcutaneously gave similar plasma concentration-time profiles of DFUR, which did not indicate prolonged release in vivo. DFUR-containing chitosan microspheres with fairly large size and good drug content could be obtained by the present preparation but remained to be improved for drug release properties.