Integration of new DNA into a cellular chromosome can alter the activity of nearby genes, sometimes affecting subsequent cell growth. A potent form of insertional mutagenesis involves integration of retroviral DNA produced by reverse transcription, a required step in the replication of retroviruses. In recent years retroviral replication has been adapted to allow new gene addition by retroviral vectors. Early in the history of retrovirus research, analysis of insertional mutagenesis in laboratory animals was found at times to result in transformation, leading to the discovery of cellular proto-oncogenes. In-depth analysis of the genetic consequences showed that integration of retroviral DNA could alter the gene activity in a variety of ways. Mechanisms of retroviral insertional mutagenesis in humans are much less well documented. However, recent work from the gene therapy and HIV fields now specify four genetic mechanisms of retroviral insertional mutagenesis in humans: (1) gene activation by integration of an enhancer sequence encoded in a retroviral vector (enhancer insertion), (2) gene activation by promoter insertion, (3) gene inactivation by insertional disruption, and (4) gene activation by mRNA 3′ end substitution. In each example, integration in patients was associated with clonal expansion or frank transformation.