Mycoplasma penetrans is a newly isolated Mollicute from the urine of patients infected with human immunodeficiency virus that demonstrates the capacity to adhere to and invade human cells. A previous report, based on assays with mouse red blood cells (RBCs), indicated that M. penetrans lacked hemolytic activity. In our studies, we incubated different isolates of M. penetrans with various RBC species and observed hemolytic zones surrounding individual mycoplasma colonies. All M. penetrans strains displayed hemolysis after 2 to 3 days of incubation. Hemolytic activity diffused from single colonies, eventually causing complete lysis. Hemolysis was most pronounced with sheep RBCs, followed by horse, chicken, and human cells. Furthermore, hemolytic activity was demonstrable in both intact mycoplasma cell preparations and spent culture supernatant. However, unlike intact mycoplasmas, the hemolytic activity in the supernatant was dependent on the reducing agent, cysteine. In addition to hemolysis, a brown precipitate was closely associated with mycoplasma colonies, suggesting oxidation of hemoglobin. Absorption spectra indicated that hemoglobin was oxidized to methemoglobin, and the addition of catalase demonstrated H₂O₂-mediated hemoxidation. Other experiments suggested that hemoxidation enhanced total hemolysis, providing the first evidence of both hemolytic and hemoxidative activities in M. penetrans.