Proviral integration is thought to be an obligate step of the retroviral replication cycle but the lentivirus visna has been reported to replicate in sheep choroid plexus (SCP) cultures in the absence of proviral integration. Because of new evidence that visna virus has a functional integrase, we reexamined visna virus infection of SCP cultures and found that proviral integration does indeed occur in this setting. While the majority of viral DNA remains unintegrated, integrated proviruses arise early in infection and accumulate over time. The sequences of the resulting host-virus DNA junctions show that, like other retroviruses, visna loses terminal nucleotides from its DNA upon integration. However, unlike other retroviruses, in over half the host-U3 junctions analyzed only a single nucleotide was lost such that the universally conserved CA dinucleotide, two nucleotides from the end of unintegrated viral DNA, did not directly abut host sequences in the provirus. We analyzed the role of integration in visna replication by introducing a series of five mutations into the integrase gene of molecularly cloned visna virus LV1-1KS1. Each mutation abolished viral replication, suggesting that integration may be an obligatory step in replication. We also documented productive infection of SCP cultures in which cell division had been blocked by g-irradiation. The ability of visna to integrate and to replicate in nondividing cells points to the possible utility of visna-based vectors for gene transfer into differentiated cells.