The mechanism of bactericidal action of phenethyl alcohol (PEA) in E. coli, which was previously demonstrated to be dependent on protein synthesis, has been investigated. Mutants resistant to PEA were selected, but the resistance observed was associated with a change in permeation. PEA effects on DNA, RNA, and protein synthesis were studied with bacteriostatic and bactericidal concentrations. Similar results (inhibition of DNA synthesis and decrease in RNA synthesis) were obtained with lethal concentrations of PEA in cells pretreated with chloramphenicol, and with bacteriostatic concentrations of PEA in unpretreated cells. The PEA intracellular accumulation reached a maximum within 4 min and was not inhibited by KCN or by 2,4-dinitrophenol. The presence of phenylacetaldehyde was demonstrated in both stationary and exponential growth phase cells exposed to PEA but not in cells pretreated with chloramphenicol. These results suggested that the bactericidal mechanism of action of PEA involves its conversion into the corresponding aldehyde.