Granulysin, a 9-kDa protein localized in human cytolytic T lymphocytes and natural killer cell granules, is cytolytic against tumors and microbes but not against red blood cells. Synthetic peptides corresponding to the central region of granulysin recapitulate the lytic activity of the intact molecule, and some peptides cause hemolysis of red blood cells. Peptides in which cysteine residues were replaced by serine maintain their activity against microbes but lose activity against human cells, suggesting their potential as antibiotics. Studies were undertaken to determine the mechanism of resistance of red blood cells to granulysin and sensitivity to a subset of granulysin-derived peptides. Granulysin lyses immature reticulocytes, which have mitochondria, but not red blood cells. Granulysin lyses U937 cells but not U937 cells lacking mitochondrial DNA and a functional respiratory chain (U937rho(o) degrees cells), further demonstrating the requirement of intact mitochondria for granulysin-mediated death. Peptide G8, which corresponds to helix 2/loop 2/helix 3, lyses red blood cells, while peptide G9, which is identical except that the cysteine residues were replaced by serine, does not lyse red blood cells. Granulysin peptide-induced hemolysis is markedly inhibited by an anion transporter inhibitor and by Na(+), K(+), and Ca(2+) channel blockers but not by Na(+)/K(+) pump, cotransport, or Cl(-) channel blockers. Although recombinant granulysin and G9 peptide do not induce hemolysis, they both competitively inhibit G8-induced hemolysis. The finding that some derivatives of granulysin are hemolytic may have important implications for the design of granulysin-based antimicrobial therapeutics.