Purified gamma haemolysin of Staphylococcus aureus was characterized in relation to the alpha, beta and delta haemolysins. The sedimentation coefficient of the gamma lysin was 2-65, somewhat higher than the S20,w values of 1-4 for freshly purified alpha lysin and 1-8 for the beta lysin. The molecular weight of gamma lysin determined by gel filtration was 45000 daltons. The pI of gamma lysin was 6-0, while that of the alpha, beta and delta lysins ranged from 8-5 to 9-6. The amino acid analysis of gamma lysin was characterized by low levels of methionine and histidine. Methionine was, however, the N-terminus, which suggested that all of the amino acid might be involved in the N-terminal group. The gamma lysin was immunologically distinct from the alpha, beta and delta lysins by quantitative precipitin tests; in Ouchterlony agar gel diffusion tests, single lines of precipitation were observed which showed no evidence of cross-reactions amongst the four haemolysins. Gamma, beta and delta lysins had no affect in mice when injected at increasing doses ranging from 0 to 100 mug. The alpha lysin killed mice, the LD50 dose being 0-60 +/- 0-12 mug, or 27 to 34 mug/kg mouse tissue. Gamma lysin was, however, lethal for guinea pigs when 50 mug quantities were injected intracradially. Gamma lysin also lysed human leucocytes and destroyed C-6 (human lymphoblast) cells.