Avian leukosis virus-derived vectors were constructed with an internal transcriptional promoter and various 3' non-coding sequences. Deletions were introduced into the downstream U3 long terminal repeat (LTR) to obtain self-inactivation of LTR-mediated transcription after one round of replication. However, 3' non-coding sequences appeared to determine not only self-inactivation of the vectors but also gene transfer efficiency. Further analysis revealed the influence of these sequences on both internal gene expression and RNA packaging. One construct permitted gene transfer while inactivating 5' LTR-promoted transcription.