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Clinical isolates of Escherichia coli sensitive and resistant to penicillin were compared in lipid composition and 14C-labelled penicillin uptake, as possible factors in resistance. Except for a slight increase in the triglyceride fraction in sensitive strains there were no qualitative or quantitative differences in the classes of extractable lipids present. Gas-liquid chromatography of the phospholipid and triglyceride fatty acids of the polar and non-polar fatty acids of the bound lipids showed that the same kinds of fatty acids were present. There was an increase of myristate in the chloroform-methanol extractable lipids of highly resistant strains accompanied by a rather general decrease of other fatty acids. Gas-chromatographic analysis of the polar-bound lipids showed an increase of the beta-hydroxydecanoic acid in the resistant strains. By studying the uptake of 14C-labelled benzylpenicillin and the crypticity of the beta-lactamase, evidence has been produced that a decreased permeability of resistant strains to penicillin cooperates with beta-lactamase to induce a high level of resistance. The altered lipid metabolism may reflect the special architectural changes in the cell wall which cause decreased permeability.