This paper examines the synergistic action of carbon dioxide and nisin on Listeria monocytogenes Scott A wild-type and nisin-resistant (Nis^r) cells grown in broth at 4°C. Carbon dioxide extended the lag phase and decreased the specific growth rate of both strains, but to a greater degree in the Nis^r cells. Wild-type cells grown in 100% CO₂ were two to five times longer than cells grown in air. Nisin (2.5 µg/ml) did not decrease the viability of Nis^r cells but for wild-type cells caused an immediate 2-log reduction of viability when they were grown in air and a 4-log reduction when they were grown in 100% CO₂. There was a quantifiable synergistic action between nisin and CO₂ in the wild-type strain. The MIC of nisin for the wild-type strain grown in the presence of 2.5 µg of nisin per ml increased from 3.1 to 12.5 µg/ml over 35 days, but this increase was markedly delayed for cultures in CO₂. This synergism between nisin and CO₂ was examined mechanistically by following the leakage of carboxyfluorescein (CF) from listerial liposomes. Carbon dioxide enhanced nisin-induced CF leakage, indicating that the synergistic action of CO₂ and nisin occurs at the cytoplasmic membrane. Liposomes made from cells grown in a CO₂ atmosphere were even more sensitive to nisin action. Liposomes made from cells grown at 4°C were dramatically more nisin sensitive than were liposomes derived from cells grown at 30°C. Cells grown in the presence of 100% CO₂ and those grown at 4°C had a greater proportion of short-chain fatty acids. The synergistic action of nisin and CO₂ is consistent with a model where membrane fluidity plays a role in the efficiency of nisin action.