Bacteria lysogenic for phage λ were infected with 32 P-labelled λ phage and DNA was extracted after incubation for various times. Based on the sedimentation properties, the [ 32 P]DNA molecules are classified into three species. The molecules of species III have the same sedimentation rate as those existing in phage particles in neutral or alkaline (pH 12·4) zone centrifugations. Those of species II, which sediment 1·13 times faster than those of species III, are composed of two components which can be separated by alkaline zone centrifugation. One component sediments at the same rate as a single strand of λ DNA and the other sediments 1·14 times faster. Those of species I sediment 1·55 and 2·2 times faster than those of species III in neutral and alkaline zone centrifugations, respectively. The molecules of species III are in a majority only at the beginning of infection; those of species II increases shortly after infection and then decrease as those of species I increase to become the major fraction. Molecules of species I were prepared from 32 P-labelled λ phage with high specific radioactivities. They were allowed to stand for various times at 4°C and analysed by zone centrifugation. Molecules which sediment at rates equal to those of species II or III were produced. Calculation shows that a single decay in a molecule of species I produces a molecule of species II, and a single double-strandbreak produces a molecule of species III. The molecules of species I have greater stability to shear than those extracted from phage particles. It can be concluded that the species III are composed of linear molecules of λ DNA; species II of circular molecules in which only one of the polynucleotides in a molecule is covalently closed; and species I of circular molecules in which both strands are covalently closed. The process of formation of the molecules of species I and its similarity to that of T4 recombination are discussed.