Cultured human monocytes released cytostatic activity upon in vitro activation with lymphokines and lipopolysaccharide. This activity was mainly due to the presence of two different cytostatic factors, termed CstF I and II, which were separated by ion-exchange chromatography. At neutral pH, CstF I bound to the weak anion exchanger DEAE-Sephacel but not to the weak cation exchanger CM-Sepharose, whereas CstF II bound to CM-Sepharose but not to DEAE-Sephacel. The molecular weights of CstF I and II as determined by gel filtration were 55,000 and 40,000, respectively. Upon chromatofocusing, CstF I behaved as if it had an isoelectric point of 5.3. Neither CstF I nor CstF II bound specifically to concanavalin A-Sepharose, indicating the absence of carbohydrate residues containing alpha-D-mannopyranosyl, alpha-D-glucopyranosyl, or sterically related components. Both factors were susceptible to inactivation by proteinase K, demonstrating their protein nature. CstF II was purified more than 3,000-fold upon chromatography on CM-Sepharose and Sephacryl S-200. Ion-exchange chromatography and chromatofocusing of CstF I removed 97% of the proteins in the monocyte supernatant, but only 15% of the activity was recovered, resulting in a fivefold purification of CstF I.