Uncoating is an essential step in viral replication cycle. Little is known about the mechanism and requirement of HIV uncoating. Using an in vitro uncoating model, we demonstrate here that the uncoating of HIV-1 was efficiently induced by lysate from activated CD4+ lymphocytes, while quiescent CD4+ lymphocyte lysate was unable to uncoat HIV-1 core. The uncoating activity was associated with an induction of in vitro reverse transcription of the viral genome. Using CD4+ lymphocytes that were arrested in cell cycle, we showed that the uncoating activity required transition of cells from G(0)/G(1a) into G(1b) stage. These results strongly suggested a requirement of cell cycle-dependent specific factors for HIV-1 uncoating. The putative HIV-1 uncoating factors could be fractionated from cell lysate by gel filtration chromatography.