Down-regulation of retroviral vector expression occurs in a number of cell types after transplantation. Although a number of vector elements have been shown to affect expression in specific experimental situations, the results can vary depending on the specific cDNA being expressed, the individual retroviral elements included in vectors, the promoter, or the inclusion of selectable markers. In previous experiments with the lysosomal enzyme beta-glucuronidase, silencing has occurred in more than 95% of transduced cells regardless of the position of the expression unit within the vector, whether a eukaryotic or viral promoter was used, whether a bacterial selectable marker gene was present or not, the target cell type, or the species of the host. It has been a consistent finding that a small number of continuously expressing cells persist for long periods after transplantation. In this study we found that deletion of all the transcriptional regulatory elements from the vector LTR, inclusion of a permissive primer binding site sequence, and use of a eukaryotic housekeeping promoter could greatly increase the number of expressing cells in fibroblast grafts in subcutaneous neo-organs and in the brain. Furthermore, the level of enzyme expression was increased five-fold on a per positive cell basis, indicating that the deleted regulatory elements were exerting a negative effect on expression in the few cells that were positive before modification of the vector. This resulted in more than a 50-fold increase in total activity compared with the previous highest expressing vector.