A plaque-forming agent arose spontaneously during cloning of Acholeplasma oculi 19L. The agent produced plaques on A. oculi 19L and A. oculi-i, but not on A. laidlawii, A. modicum, or wild isolates of A. oculi. The agent required horse serum for plaque formation as well as for adsorption to the indicator lawn; however, it was extremely sensitive to an inhibitor in some horse sera. The agent retained infectivity after passage through a 50 nm filter and was heat-, Nonidet P-40-, and chloroform-labile, but relatively ether-stable. It was not determined whether the agent is a virus or a bacteriocin-like substance.