In vivo and in vitro antitumor activities of macromolecular prodrugs of doxorubicin (DXR) using 6-O-carboxymethylchitin (CM-chitin) as a carrier polymer were investigated. In order to determine the effect of a spacer group between a drug and a carrier polymer on the antitumor activity, we employed two different spacer groups, a lysosomal digestible tetrapeptide (Gly-Phe-Leu-Gly) and a simple hydrophobic pentamethylene to immobilize DXR molecules to CM-chitin. The cytotoxic activity of the conjugates obtained were investigated against four kinds of tumor cell lines, p388D₁ lymphocytic leukemia, L1210 leukemia, HLE human hepatoma and Hela utrocervical carcinoma cell in vitra The CM-chitin/Gly-Phe-Leu-Gly/DXR conjugate 1 with lysosomal digestible tetrapeptide spacer groups showed slightly higher in vitro cytotoxic activity against these tumor cells than CM-chitin/C₅/DXR conjugate 2 with a simple pentamethylene spacer group. However, the cytotoxic activity of these two conjugates was weaker than that of the free DXR. The survival effects of these conjugates were investigated against p388 lymphocytic leukemia bearing mice in vivo by intraperitoneal (i.p.) implantation and intravenous (i.v.) injection. Although these conjugates did not show very high cytotoxic activities in vitro compared with the free DXR, conjugate 1, with the lysosomal digestible tetrapeptide spacer groups, exhibited survival effects against p388 lymphocytic leukemia in mice i.p./i.v. However, conjugate 2, with the simple pentamethylene spacer group, did not show any survival effects under the same conditions. These conjugates did not display an acute toxicity in the high dose ranges.