Retrovirus vectors can be made in the absence of helper virus by using retrovirus packaging cell lines. Helper-free virus is critical for a variety of gene transfer studies. The most useful packaging cell lines contain helper virus DNA from which the signal required for packaging of the viral RNA genome into virions has been deleted. However, we showed that the ability to package virus is conferred at very low frequency to cells infected with virus from these packaging cell lines, presumably by low-frequency transmission of the deleted virus genome. In addition, these packaging cell lines can interact with some retroviral vectors to yield replication-competent virus. We constructed packaging cell lines containing helper virus DNA that had several alterations in addition to deletion of the packaging signal. The new packaging cells retained the useful features of previously available lines but did not yield helper virus after introduction of any of the vectors tested, and transfer of the packaging function was not detected.