A study has been made of intracellular P22 DNA under conditions that favor lysogeny. After infection with radioactively labeled phage, a large fraction of the linear parental DNA is converted into a fast-sedimenting form that exhibits the properties of a covalently closed circular duplex or superhelix. These properties include an elevated sedimentation rate in strand-separating solvents and the production of a slower-sedimenting circular molecule after treatment with pancreatic DNase. The closed circular molecules were purified on the basis of their ability to renature spontaneously following alkaline denaturation. The superhelical nature of this DNA was confirmed by subsequent examination in the electron microscope and the screw-sense of the tertiary turns was shown to be right-handed. A second, less abundant form of P22 DNA appeared to consist of two inter-locked superhelical molecules. The kinetics of the incorporation of [ 3H]thymidine reveal that DNA synthesis precedes the formation of superhelical DNA molecules; most of these molecules are formed after DNA synthesis ceases. Superhelical molecules seem to be efficiently formed from both parental and newly made DNA. The number of phage equivalents of newly synthesized, superhelical DNA was estimated as 10 to 20 per cell under the conditions used.