Premature termination products of reverse transcription that consist physically of viral minus-sense single-stranded DNA that is shorter than one long terminal repeat and partial DNA duplexes are dramatically increased in the central nervous system (CNS) of FVB/N mice that are infected by ts1, a temperature-sensitive mutant of Moloney murine leukemia virus. Due to their migration in agarose gels, these incomplete physical forms of DNA have been designated high-mobility (HM) DNA. In non-CNS tissues, the level of HM DNA is either low or not detectable. In order to determine the conditions that are necessary for the synthesis of HM DNA in vivo, we have characterized the physical forms of HM DNA that were synthesized in vitro in chronically infected SC-1 cells after temperature upshift. At the permissive temperature of 34°C, the chronically infected SC-1 cells did not synthesize HM DNA. After temperature upshift of the cultured cells from 34 to 37°C, the chronically infected SC-1 cells developed extremely high levels of HM DNA. Following temperature downshift of the cultured cells from 37 to 34°C, a decrease in the level of HM DNA and an increase in the level of unintegrated linear proviral DNA occurred simultaneously. These results suggested that the accumulation of HM DNA both in vitro and in vivo may be the result of superinfection.