An investigation of Klebsiella pneumoniae hemolytic activity was carried out. Strains isolated from different infected specimens were hemolytic in tryptic soy agar with rabbit blood; incubation at 4 °C enhanced the hemolysis. There was no evident red blood cell lysis in plates with human, sheep, mouse, and chicken erythrocytes. The culture in tryptic soy broth, its supernatant and bacterial lysate did produce evident hemolysis of rabbit red blood cells when they were preincubated with 2-mercaptoethanol. Klebsiella pneumoniae hemolysin showed the Arrhenius effect, while temperatures over 60 °C for 10 min reduced the activity of crude hemolysin to zero; purified hemolysin, however, was heat stable. Two hemolysins active on rabbit red blood cells were purified and both shared several properties. This work represents the initial description of a thiol-activated hemolysin in Gram-negative bacteria.