The morphological changes in Serratia marcescens induced by astromicin were determined by a new technique of electron microscopy, a rapid freezing and substitution fixation technique, and a freeze-fracturing technique. Two structural changes were observed. One was damage to the cytoplasmic membrane, and the other was the accumulation of a large electron-dense mass in the cytoplasm. The damage observed in the cytoplasmic membrane was the disappearance of the unit membrane structure from the thin-sectioned profile of the drug-treated bacteria and the loss of the membrane particles from the fractured surface of the membrane. Damage to the membrane was also suggested by the results of examination of the spheroplasts for stability. The spheroplasts prepared from the drug-treated bacteria were unstable in an osmotically controlled buffer. Most of the spheroplasts were lysed within 3 h, whereas those prepared from control cells were stable for more than 15 h. The electron-dense mass in the cytoplasm was usually seen in the polar region of the cell in close contact with the cell membrane. These structural changes were not specific for astromicin but were also found in gentamicin-treated cells.