Previous work has indicated that β-diethylaminoethyl-diphenylpropylacetate (SKF 525-A), a well-known inhibitor of microsomal drug-metabolizing enzymes, lowers surface tension and has a biphasic hemolytic effect on the human red blood cell membrane. Present studies further investigate the effect of SKF 525-A on biological membranes and are concerned with the action of SKF 525-A on bacterial cells. A diploid mutant of Escherichia coli which has limited ability to synthesize pyrimidines due to the absence of orotidylate decarboxylase was used for these experiments. SKF 525-A at high concentrations (10 −2 and 10 −3 M) lysed E. coli (diploid mutant) cells either during the exponential or secondary growth phase. However, the rate of cell lysis was greater during the exponential than during the secondary growth phase. The magnitude of cell lysis in both growth phases correlated well with surface activities of SKF 525-A and was linear during the first 30 min. E. coli cells treated with SKF 525-A (10 −5 M) for 4 hr prior to incubation with either uracil or L-isoleucine demonstrated decreased cellular accumulation of these substrates. The degrees of inhibition by SKF 525-A on the cellular uptake of these substrates were similar in spite of the dissimilarity of structures. The phenomena of cell lyses and impaired uptake of metabolic substrates suggest that the effect of SKF 525-A concerns an alteration of the cell wall and/or cell membrane rather than a specific effect on transport mechanisms.