Integration into the host-cell genome is a critical step in the retrovirus life cycle. In particular, the choice of the integration site is crucial for retroviral replication, since integration at a site incompatible for high-level transcription may impair production of the progeny virus. Integration is not sequence specific, thus all chromosomal sites could potentially host integration events. However, this is not what is observed in vivo, where integrated viruses are preferentially detected in chromatin regions characterized by an open structure, a hallmark of actively transcribed genes. Target site selection might be influenced by several factors, including the function of cellular proteins that interact with integrase, the viral protein that catalyzes the integration reaction. Interestingly, a common functional feature that unifies these cellular co-factors is that, to a different extent, they are all involved in the regulation of chromatin structure or transcription. Inappropriate retroviral integration might lead to insertional mutagenesis and cellular transformation, as recently observed in a gene therapy clinical trial exploiting retroviral vectors for gene transfer into hematopoietic progenitors. Thus, the deeper understanding of the molecular mechanisms regulating integration site selection is also essential for the design of safer and more effective gene transfer vectors.