The growth of minute virus of mice (MVM) in L cells was followed by plaque assay of both cell-associated and free virus at intervals up to 36 h after infection. The major production of progeny virus occurred after incubation for 27 h and most of the virus remained cell-associated. L cells infected with MVM were pulse-labeled with 3H-thymidine for 6 h preceding induction of lysis with sodium dodecylsulfate. Two new species of DNA remained in the supernatant fractions from lysates after selective precipitation by 1 M NaCl of cellular DNA. These first appeared 17 h after infection. On the basis of sedimentation rates and response to heating and to treatment with alkali, one of these species is a hydrogen-bonded duplex molecule, which on denaturation hybridized with the single-stranded DNA of MVM virions.