Cells of the human erythroleukemia cell line K562 constitutively secrete a factor that inhibits human T lymphocyte proliferation induced via CD3/Ti. The factor, termed K-TIF (K562-derived T cell inhibitory factor) is produced in either the presence or absence of fetal calf serum in cultures of K562 cells and can be precipitated by 70% NH4SO4. Gel filtration chromatography on Superose 12 resin by FPLC showed that the inhibitory factor has a molecular weight of approximately 30-35 kDa. A protein of this size, metabolically labeled with [35S]methionine, specifically bound human peripheral blood mononuclear cells. Chromatofocusing with Mono P by FPLC (pH gradient 7.2-5) indicates that the inhibitory factor has an isoelectric point of 6.0-6.4.