The characteristics of hemolysis produced by streptolysin O (SLO) were investigated in rabbit, human, and rat erythrocytes. Kinetic studies of hemoglobin (Hb) release showed that rabbit and human erythrocytes exhibited typical "multi-hit" survival curves. Extrapolation of these curves to the ordinate indicated that approximately two molecules of SLO may be sufficient to produce lysis of a single cell. In contrast, exponential ("single-hit") survival curves were observed when rat erythrocytes were treated with SLO. At 0 C, high concentrations of SLO rapidly lysed rabbit erythrocytes; low concentrations had no effect on the cells at this temperature. The release of intracellular (86)rubidium ((86)Rb(+)) and Hb in rabbit erythrocytes exposed to SLO was investigated. In the presence of phosphate-buffered saline, rubidium and Hb were lost at the same rate from toxin-treated cells. The addition of bovine serum albumin to the suspending medium did not retard the escape of Hb, and the efflux of (86)Rb(+) only slightly preceded Hb loss. Addition of sucrose to the cells delayed both Hb and rubidium release. These results are interpreted as indicating that the "colloid-osmotic" lytic process is not involved in the hemolysis of erythrocytes by streptolysin O.