A carcinostatic protein fraction (EPF) was separated from Escherichia coli 055 cells by the following procedure: E. coli 055 cells were suspended in distilled water, and extracted by repeater freezing and thawing. The extracts were centrifuged and the supernatant was treated with 1N acetic acid (pH 4.5). Acid precipitate collected by centrifugation was dissolved in alkaline water (pH 8 to 9) and subjected to gel filtration on Sephadex G-200 column. The high molecular weight fraction obtained by gel filtration was collected, freeze-dried, and designated as EPF. EPF was composed of approximately 74.5% of protein, 20.4% of lipid, and a small amount of polysaccharide. The isoelectric point of EPF was pH 5.2 +/- 0.A and the ultraviolet spectrum showed absorption maximum at 278 nm (E1%1cm 4.30) in 0.01M Tris-HCl (pH 7.8) buffer. The antitumor experiment with EPF demonstrated that the daily injection of EPF in a dose of 10 microgram/mouse inhibited sarcoma-180 ascites in 90% of the treated animals and the dose of 100 micrograms/mouse brought about 100% inhibition. In the contact test using C3H2K and C3HW2K cells, 200 micrograms/ml of EPF exhibited about 40% cell destruction. In the ihhibition test of 14C-phenylalanine incorporation into intact sarcoma-180 cells, EPF exhibited 60.5% inhibition at the concentration of 275 micrograms/ml. In contrast, EPF did not show any inhibition of the incorporation of 3H-uridine or 3H-thymidine.