Infectious virus nucleic acids were extracted from the Hirt supernatant of chicken embryo fibroblasts infected for different times with the Schmidt-Ruppin strain of Rous sarcoma virus (RSV). Infectious DNA and DNA-RNA hybrid molecules could be recovered from 6 h after infection in experiments using 1.5 X 10(9) infected cells. Only small amounts of infectious virus DNA could be purified 6 h after infection whereas at 24 h approximately one infectious DNA molecule could be recovered for each input virus infectious unit. At 24 h, both infectious supercoiled and non-supercoiled molecules were found. The specific infectivity of the supercoiled fraction was less than that of the non-supercoiled fraction. Infectious supercoiled DNA could be recovered from 16 h after infection. Evidence is presented that both forms of unintegrated virus DNA may rest unintegrated for at least 8 days in the cell, though chronically infected cells were shown to contain less than one unintegrated molecule per 10(2) to 10(3) cells.