Endostatin, a carboxy-terminal fragment of collagen XVIII, has been shown to act as an anti-angiogenic agent that specifically inhibits proliferation of endothelial cells and growth of various primary tumors. Here, we describe the expression by Chinese hamster ovary (CHO) cells of murine endostatin and of a tagged-fusion protein, (his)6-met-endostatin. A dicistronic mRNA expression vector was utilized in which endostatin cDNA was inserted upstream of the amplifiable marker gene, dihydrofolate reductase (DHFR). After transfection of the expression vectors, stepwise increments in methotrexate levels in the culture medium were applied, promoting gene amplification and increasing expression levels of the proteins of interest. The expression level of secreted native endostatin was about 78 mug/mL while the one for secreted (his)6-met-endostatin was about 114 mug/mL, for the best expressing clones. Characterization of physico-chemical and immunological activities of the proteins was performed using SDS-PAGE and Western blotting. The biological activities of recombinant endostatins were tested with a cow pulmonary artery endothelial (C-PAE) cell proliferation assay. Both recombinant endostatin and (his)6-met-endostatin inhibited, in a dose-dependent fashion, growth of C-PAE cells stimulated by basic fibroblast growth factor (bFGF). (C) 2004 Elsevier Inc. All rights reserved.