Earthworm coelomocytes have been used as effector cells against the human tumor target, K562. To first assess the viability of effectors, incorporation of [3H]-thymidine was tested and was higher in autogeneic (A<==A, self) than in allogeneic (A<==>B, nonself) coelomocytes. A<==>A showed significantly greater numbers in S, G2, or M phases than A<==>B coelomocytes. When A<==>A or A<==>B were cultured, no significant cell killing occurred in either, as measured in a 4-hr 51Cr release assay. A<==>A but not A<==>B killed K562 target cells. Cytotoxicity was dependent upon membrane binding between small, electron-dense coelomocytes and targets; it was enhanced by adding PHA. The heat labile supernatant from A<==>A but not from A<==>B killed K562 targets after cultivation for 10 min at 22 degrees C, but not immediately after washing. Recognition of, binding to, and killing of foreign cells in a natural killer cell-like reaction may reflect natural immunity in earthworms.