This study investigates the effects of N-(n-dodecyl)diethanolamine (DDA) on enzymes and growing cells of Escherichia coli NCIMB 8277. Enzyme activities in the presence of DDA were determined by measuring substrate-dependent oxygen consumption by whole cells, or of NADH formation or oxidation by cell extracts. Lysis of growing cells was followed by measuring changes in turbidity and cell count. DDA promptly arrested oxygen uptake on pyruvate and acetate, due to cofactor loss rather than to enzyme denaturation, since cell-free glyceraldehyde-3-phosphate and NADH dehydrogenases remained active. Formate and succinate oxidation by membrane-bound enzyme systems independent of cofactors was likewise unaffected. DDA lysed growing cells at rates related to drug concentration, pH, and the previous growth rate. Loss of cellular enzyme activity following addition of DDA is due to cofactor leakage and not to enzyme denaturation. Whereas nongrowing cells remain intact in the presence of DDA, actively-growing organisms undergo lysis, consistent with autolysin action. Cell lysis, not normally observed with membrane-active antimicrobials, also occurs with cetrimide, and may be dependent on the alkyl chain length in these compounds. The action on growing cells parallels that of penicillin and daptomycin, which bears a decanoyl residue that penetrates the cell membrane, causing leakage and membrane depolarization.