Human immunodeficiency virus (HIV)-based vectors are being increasingly used in vitro for gene transfer and in vivo for gene therapy. The proportion of integrated retroviral vectors that are silenced or remain transcriptionally active, and the stability of gene expression in the latter remains poorly explored. To study this, T cells were infected with an HIV-1-based vector construct containing a long terminal repeat-driven reporter gene. Only a small percentage of detectable integrated vector expressed gene product. In clones derived from cells with transcriptionally active vector, gene expression was remarkably stable with more than 80% continuing to express for greater than 18 months. Failure to continue expressing the vector was associated with epigenetic changes. Our data suggest that there are two forms of vector silencing: one occurring immediately after integration affecting the majority of the vectors, and one occurring in the much longer term affecting a small minority of vectors which had previously established expression.