Although Mycoplasma pulmonis did not lyse normal erythrocytes, it rapidly lysed erythrocytes that had cytoskeletal deficiencies which allow increased diffusion of membrane glycophorin or that had been treated with trypsin to remove surface proteins. This hemolysis occurred only in the presence of bovine serum albumin and was eliminated by trypsin treatment of the mycoplasma. Hemolytic activity was restored after such trypsin treatment when mycoplasma protein synthesis was allowed. M. pulmonis hemolytic activity was not diffusible and thus differed from the activities reported for other mycoplasmas, which involve small diffusible intermediates such as hydrogen peroxide. With the exception of the requirement for bovine serum albumin, the factors which affected hemolysis were similar to those which we have previously reported to affect M. pulmonis hemagglutination, suggesting that these two activities are functionally related.