A factor produced by lipopolysaccharide-stimulated human monocytes, monocyte-derived eosinophil cytotoxicity-enhancing factor (M-ECEF), increases the ability of human eosinophils to kill larvae of Schistosoma mansoni. In order to purify this monokine, a continuous cell line was sought as a generator of source material. It was found that high titers of an ECEF-like activity could be obtained from the U937 cell line cultured in serum-free medium. Production of this activity was optimal when cells were cultured with PMA for 2 days and were further treated with LPS for 2 days. PMA and LPS alone did not enhance eosinophil cytotoxicity and could be separated completely from U937-ECEF activity by reversed-phase HPLC. Thus, the activity was not due to carry-over of these two stimuli. U937-ECEF was compared with M-ECEF by a number of analytical methods. ECEF from both sources was resistant to several denaturing treatments but was sensitive to proteases or to reduction and alkylation. U937-ECEF exhibited activity profiles similar, if not identical, to those of M-ECEF when subjected to molecular sizing HPLC in the presence of 8 M urea, isoelectric focusing, and reversed-phase HPLC. The activity has apparent m.w. of 17,000 and 32,000, isoelectric points ranging from 3.8 to 5.1, and one or more reversed-phase HPLC retention times, depending on the method of sample preparation. These results demonstrate certain physical characteristics of M-ECEF, show that the U937 cell line is an appropriate source for the purification of M-ECEF, and provide information that will allow the design of a purification strategy. Although it appears that tumor necrosis factor (TNF) or a TNF-like molecule is a component of M-ECEF, a major component of M-ECEF is different from TNF as judged by the 1) physical characteristics of M-ECEF, 2) low direct toxicity of M-ECEF to L929 cells, 3) comparative stability of M-ECEF to heat treatment, and 4) inability of an anti-TNF monoclonal antibody to remove M-ECEF activity.