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To delineate parameters contributing to the extracellular lifetime of retroviral vectors, we carried out stability tests of retroviruses derived from cell lines of different origin and kept under different cultivation conditions. Results show that amphotropic mouse retroviruses (MLV-A) derived from human and hamster cells exhibit 2- to 3-fold higher half-lives compared to retroviruses from mouse cells. Cultivation at 32°C has been reported to yield high virus titers. However, the benefit of virus production in mouse cells at 32°C is controversial. In our hands the cultivation temperature affected, hitherto not noticed, the half-life time of MLV-A. The 37/32°C shift resulted in a 3-fold decrease of viral half-lifes compared to MLV-A released from mouse cells at 37°C. Thus, MLV-A released at 37°C is phenotypically different from MLV-A synthesized at 32°C. Increased virus stability was inversely correlated with the level of cholesterol in the viral membrane. Finally, depletion of viral cholesterol in vitro resulted in intact virus with increased thermal stability. Thus, retrovirus lability depends on the host cell and parallels the cholesterol amount in the viral lipid shell. [Copyright &y& Elsevier]