Marked cytopathic effect was produced in both porcine lung fibroblasts and MRC-5 human lung fibroblasts with doses of 7.5 X 10(6) and 1.5 X 10(7) CFU of Mycoplasma hyopneumoniae J per ml, respectively. M. hyopneumoniae VPP11, at a concentration as low as 1.25 X 10(7) CFU/ml, was capable of causing cytopathic effect in MRC-5 monolayers also. The infected monolayers developed patches of detached or lysed cells within 48 h. At a protein concentration of 2 micrograms/ml, M. hyopneumoniae membranes, prepared from freeze-thaw lysed cells, were capable of inducing cytopathic effect in MRC-5 fibroblasts within 24 h. The cytoplasm did not contain any cytotoxic activity. This indicates that the cytopathic factor is located in the cell membrane. This factor appears to be protein in nature. It was heat labile (100 degrees C, 15 min) and was destroyed by pronase (1 mg of pronase per 1 mg of membrane protein). It appears to be antigenic, since rabbit anti-M. hyopneumoniae serum was capable of neutralizing its cytopathic activity.