Mouse leukemia cells and normal thymocytes bearing thymus-leukemia (TL) cell surface antigens were previously shown to acquire resistance to lysis by guinea pig complement (C) during incubation with TL alloantiserum in vitro at 37 degrees C due to heat-labile serum activity resulting in deposition of mouse C3 onto the cell surface. The role of heat-labile serum activity and C3 in modulation of TL+ cells in vivo in mice actively or passively immunized against TL antigens was investigated. Mice of the TL-/TL+ C57BL/6J (B6) strain and the B6 congenic strain B6-Tiaa possessed poorly modulating sera, and the radiation-induced A-strain leukemia RADA1 transplanted into B6 mice passively immunized with heated (56 degrees C) TL antiserum failed to modulate; thymocytes of B6-Tiaa mice immunized similarly also did not modulate. A specific requirement for mouse C3 deposition onto RADA1 cells to achieve a modulated state was demonstrated in actively immunized TL- (B6 X A-Tiab)F1 mice in which circulating C3 and modulating activity were depleted by administration of cobra venom factor. In immunized (B6 X A-Tiab)F1 mice bearing RADA1 transplants and repeatedly given injections of B6 serum, tumor cells escaped immune destruction despite a lack of modulation. Thus modulation of TL antigenicity on tumor cells in vivo, but not tumor escape, required cell-bound C3.