Retroviral vectors (RVs) have been used for stable gene transfer into mammalian cells for more than 20 years. The most popular RVs are those derived from the Moloney murine leukaemia virus (MoMLV). One of their main limitations is their inability to transduce noncycling cells. However, they have a relatively simple genome and structure, are easy to use, and are relatively safe for in vivo applications. For the last two decades, the artificial evolution of RVs has paralleled evolution in their applications, which now include those as diverse as the generation of transgenic animals, the stable delivery of small interfering RNA (siRNA) and gene therapy clinical trials. Recent reports of two successful gene therapy clinical trials in patients with severe immunodeficiency disease in France and Italy, and the development of T-cell acute leukaemia in two of 10 children participating in one of these clinical trials, demonstrate the great potential of RVs, but also some potential risks which may be intrinsically associated with their use. Basic aspects of RVs and vector production were reviewed in detail in a previous supplement of this journal. This article will first summarize some general aspects of retroviruses and RVs. Thereafter, recent developments in gene therapy using RVs, novel applications such as stable RNA interference and some other recent issues related to retroviral integration, including clonality studies after haematopoietic stem cell transplantation, retroviral tagging and insertional oncogenesis will be discussed.