The present invention relates to a method of producing covalently closed circular (ccc) DNA from a cultured cell or unicellular organism, preferably a bacterium, essentially free of genomic DNA. The method of the invention comprises the steps of precipitating a cleared lysate with an alcohol, washing and resuspension of the precipitate, digestion of the genomic DNA with a nuclease that cleaves linear DNA or circular DNA that includes a nick or a free 3′ or 5′ end but not ccc DNA, and finally, separation of the purified ccc DNA from the remainder of the product of the digestion step by contacting the product with an ion exchange material. ccc DNA obtained by the method, pharmaceutical compositions comprising the ccc DNA, and a kit for carrying out the method of the invention are also contemplated.