Retroviral vectors that are able to sustain multiple rounds of replication may find many applications. However, one critical feature of such vectors is the ability to maintain an intact transgene cassette during repeated rounds of replication. We here report on the stability of a translational cassette consisting of an internal ribosome entry site followed by the enhanced green fluorescent protein coding sequence inserted in different configurations into murine leukemia virus genomes. In two of the constructs, the insert was located in the upstream part of the U3 region while in the third construct it was inserted in the 3' untranslated region of the viral genome. Furthermore, in two of the constructs, the translational cassette was flanked by a direct repeat, while no such structure flanked the third construct. Our results show that deletion of the heterologous translational cassette is observed for all constructs upon extended cell culture and that the number of replication rounds before revertants are detected can be postponed by decreasing the length of the repeat flanking the translational cassette.