An inhibitory factor, which has been shown to suppress the uptake of 125I-iododeoxyuridine by both lymphoid and nonlymphoid cells, was isolated from the supernatant of an Epstein-Barr virus- (EBV) transformed B cell line (1605L) established from a cotton-topped marmoset. Purification of the inhibitor, which was produced in serum-free medium by crowded cultures of the 1605L cells, was achieved by DEAE-cellulose chromatography followed by preparative polyacrylamide gel electrophoresis. The apparent m.w. of the 1605L factor was determined to be 65,000 to 70,000 by SDS-polyacrylamide gel electrophoresis. The inhibitor was sensitive to digestion by trypsin and chymotrypsin but not RNase or DNase, indicating that it was protein in nature. Exposure of the 1605L factor to 56 degrees C for 1/2 hr or pH 2 for 48 hr at 4 degrees C destroyed its inhibitory activity. The biochemical characteristics and activity of the 1605L inhibitor distinguish it from Type I interferon and several other soluble immunologic mediators known to be produced by lymphoid cell lines.