The mode of action of difficidin (DIF) was investigated. Upon addition of DIF to log phase cultures of Escherichia coli, growth ceased immediately and small round cells accumulated after 30 minutes incubation. No cell lysis was observed. DIF was rapidly bactericidal to both growing and stationary phase cultures, and inhibited protein synthesis more rapidly than RNA, DNA, or cell-wall synthesis in growing cells. Protein synthesis was also inhibited in a cell-free system. The frequency of natural mutation to resistance in E. coli was less than 1 in 10¹⁰ cells.