After fusion of the human immunodeficiency virus type 1 (HIV-1) envelope with the host cell membrane, the HIV-1 core enters the cell cytoplasm. Core components are then restructured to form the reverse transcription complex (RTC); the biochemical details of this process are currently unclear. To investigate early RTC formation, we characterized the endogenous reverse transcription activity of virions, which was less efficient than reverse transcription during cell infection and suggested a requirement for a cell factor. The addition of detergent to virions released reverse transcriptase and capsid, and reverse transcription products became susceptible to the action of exogenous nucleases, indicating virion disruption. Disruption was coincident with the loss of the endogenous reverse transcription activity of virions, particularly late reverse transcription products. Consistent with this observation, the use of a modified "spin thru" method, which uses brief detergent exposure, also disrupted virions. The addition of lysates made from mammalian cell lines (Jurkat, HEK293T, and NIH 3T3 cells) to virions delipidated by detergent stimulated late reverse transcription efficiency. A complex with reverse transcription activity that was slower sedimenting than virions on a velocity gradient was greatly stimulated to generate full-length reverse transcription products and was associated with only relatively small amounts of capsid. These experiments suggest that cell factors are required for efficient reverse transcription of HIV-1. Copyright 頲008, American Society for Microbiology. All Rights Reserved.