The mechanism of action of vibriocin, a bacteriocin produced by Vibrio comma, was investigated. Its lethal action (as defined by the loss in colony-forming ability) was reversed by tryptic digestion within 7 to 10 min after adsorption. The bacteriocin had a pronounced inhibitory effect on deoxyribonucleic acid (DNA) synthesis, whereas ribonucleic acid (RNA) and protein synthesis continued, although at a reduced rate. Chloramphenicol protected sensitive bacteria from the lethal action. Degradation of bacterial DNA prelabeled with (3)H-thymidine, as measured by changes in acid-precipitable radioactivity, occurred 10 min after treatment with vibriocin. The bacteriocin per se had no detectable deoxyribonuclease activity. Observation of vibriocin-treated cells by phase-contrast microscopy, measurement of ultraviolet light-absorbing capacity of extracellular fluid, and (42)K-efflux studies indicated a damaged bacterial membrane. This impairment of membrane function occurred in the presence of chloramphenicol and thus, unlike the lethal effect of vibriocin, was independent of protein synthesis.