A lambda DNA supercoil system has been developed to study the effects of colicin E2 on DNA in vivo. Colicin E2, a protein antibiotic synthesized by strains of coliform bacteria that carry the Col E2 plasmid, had as its most conspicious effect damage to the DNA of sensitive strains. Colicine E2 attacks the supercoiled molecul formed by labeled lambda DNA in superinfected cells as well as it attacks the bacterial DNA. The rate and extent of acid solubilization of the lambda supercoils and of host bacterial DNA induced by E2 treatment are nearly identical. Treatment of superinfected cells with colicin E2 results in the progressive conversion of lambda DNA supercoils to open circles and/or linear full lenght molecules, and subsequently to fragments less than full lambda in size. The first endonucleolytic reactions are single-strand and or double-strand breaks. The rate of supercoil breakdown as well as the final percent supercoils remaining unconverted, the size of the final lambda fragments, and the extent of solubilization are dependent on the multiplicity of colicin used. Additions of trypsin to E2-treated superinfected cells results in a cessation of further breakdown of the lambda molecules, presumably as a result of digestion of accessible colicin molecules. Energy is essential for an early event in colicin E2 action. The host enzymes, endonuclease I and Rec BC, may be instrumental in the nucleolytic process caused by colicin E2: endonuclease I in reaction preceding cell killing and Rec BC in a secondary degradation of the bacterial DNA.