The 65 kD homologous restriction factor (HRF) was isolated from normal human erythrocytes (E) by immunoadsorption using rabbit anti-human HRF. The protein was radiolabeled and incorporated into the membrane of sheep erythrocytes (Es). Es bearing HRF exhibited a markedly reduced susceptibility to reactive lysis by C5b-9. Es-HRF with 1,000-3,000 HRF molecules per cell and sensitized with rabbit IgG anti-Es also were less susceptible to lysis by human large granular lymphocytes (LGL) than untreated Es sensitized with IgG antibody. Similarly, human E of a patient with paroxysmal nocturnal hemoglobinuria (PNH), lacking HRF and sensitized with IgG antibody underwent lysis by human LGL. Lysis was abrogated by incorporation of isolated human HRF. Incorporation of human decay-accelerating factor (DAF) into sensitized Es had no effect on antibody-dependent, cell-mediated cytotoxicity. Furthermore, lysis of Es by the isolated cytolytic C9-related protein (C9RP) of human cytotoxic lymphocytes could be inhibited by cell bound human HRF. These results suggest that HRF inhibits channel formation not only by C5b-9, but also by cytotoxic lymphocytes.