<span class="paragraphSection"><div class="boxTitle">Abstract</div>Retroviral integration is mediated by viral integrase (IN), which synapses two viral long terminal repeat DNA ends and produces a series of nucleoprotein complexes known as intasomes. While structural studies of mature intasomes have illuminated key aspects of their architecture and provided insights into the integration reaction, the sequence of events driving IN oligomerization and engagement of the viral DNA pairing remains unclear. Here, using complementary biochemical and biophysical approaches, including ensemble and single-molecule Fӧrster resonance energy transfer, we reveal that integration progresses through a key transient intermediate that leads to the mature intasome. We demonstrate that Rous sarcoma virus intasome assembly pathway proceeds through a tetrameric intermediate where two IN dimers engage a single DNA end. This complex subsequently oligomerizes to form mature, functional octameric intasome in which two DNA ends are juxtaposed for concerted integration. These findings provide mechanistic insights into the stepwise pathway of retroviral integration and define a previously uncharacterized intermediate critical for intasome maturation, and possibly a drug target for clinically relevant retroviruses.</span>