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Obtaining high expression levels of a therapeutic gene in target cells could be achieved by integrating multiple copies of a recombinant retrovirus. However, we observed that cells retrovirally infected at high multiplicities of infection (MOIs) carried only single or double integrated proviral copies, suggesting that maximum retroviral transduction was achieved at relatively low MOIs. The same results were obtained when purified virus, free of most medium components, was used. Retroviral infection was shown to be inhibited by supernatants of other viral producer cell lines, and this inhibition could be removed by a centrifugation step that also removed more than 90% of infectious virus. Quantitative-competitive PCR of retroviral preparations showed that the amount of retroviral vector RNA present was similar to the amount expected on the basis of virus titers. Our data suggest that retroviral preparations derived from PA317 packaging cells contain inhibitors that copurify with retroviruses and do not contain viral vector RNA. We postulate that these inhibitor particles cannot achieve a productive infection but interfere with transduction of the target cells by infectious virions. This study might define an important criterion for the selection of more effective packaging cell lines.