The effect of leukocyte extract on the viability of certain human Mycoplasma was investigated. Leukocytes were obtained from venous blood of human donors or from glycogen-induced peritoneal exudate of rabbits. The cells were suspended in citrate buffer at various pH values and the extract was prepared by repeated quick-freezing and thawing. The supernatant of these preparations showed a marked mycoplasmacidal effect on M. pneumoniae, M. salivarium and M. fermentans. Fractionation with ammonium sulfate revealed that the activity of this extract resides in the fraction obtained at 40% to 60% saturation of ammonium sulfate. This mycoplasmacidal factor exhibits optimal activity at about pH 4.7, is non-dialyzable and is relatively heat resistant.