Analogous to transmission of human T-cell leukemia virus type 1 (HTLV-1) in vivo, an in vitro cell-to-cell infection model was established by coculturing MT-2 cells as virus donors and HUT78 cells as recipients. At a donor:recipient ratio of 1:2, cell fusion occurred and a new round of HTLV-1 genome replication was initiated in the cocultured cells. Newly synthesized unintegrated viral DNA was detected by Southern blot within 4-8 h and then increased between 8 and 48 h following cell mixing. The most dominant species of unintegrated viral DNA was 3.7 kb in size which hybridized to a full-length HTLV-1 DNA probe but not to a Kpnl viral DNA fragment that is absent from a defective proviral genome that has been previously identified in MT-2 cells. Northern blot analysis showed large amounts of viral RNA in the virus donor cells and in the cocultured cells, with a 3.4-kb species being the most abundant. This 3.4-kb RNA gave a pattern identical to that of the 3.7-kb unintegrated viral DNA in hybridization studies using the two probes. It seems likely that the unspliced RNA transcript from the defective proviral genome in MT-2 cells was effectively reverse transcribed upon initiation of cell-to-cell viral transmission to susceptible HUT78 cells. Despite active de novo reverse transcription, however, viral RNA levels remained unchanged following cell-to-cell transmission of HTLV-1 infection and no viral antigen production could be attributed to the newly initiated round of viral genome replication. As an abortive infection model this simple cell-to-cell infection system warrants more detailed study as it has the potential to provide reliable information regarding the early events in HTLV-1 transmission and infection.