As a possible consequence of their survival strategy, proviruses are predominantly found in transcription-promoting genomic sites. For certain applications, these findings have led to the preferential use of retroviral vectors for the stable integration of transgenes. This study demonstrates that transcription levels of single-copy proviruses, which have been established either by infection or by single-copy transfection (electroporation), are rather comparable. Therefore, electroporation is suggested as an alternative gene transfer route in cases where the use of infectious retroviral vehicles is to be avoided due to safety considerations. A difference between clones derived from these two gene transfer routes concerns the inactivation pattern which, for electroporated clones, is an exclusive property of the low expressers. This difference may be due to the nature of the illegitimate recombination event which is thought to be less invasive if catalyzed by the retroviral integrase. Substantial differences between infection and Ca phosphate-mediated transfection that have been reported earlier are explained by the respective transfection parameters.