Transmission of human T cell leukemia virus type I (HTLV-I) to a T cell line (MOLT-4#8) was studied using cell-free virus infection or cocultivation with an HTLV-I-transformed T cell line (MT-2). Immunofluorescence and FACS analyses showed that HTLV-I was efficiently adsorbed onto MOLT-4#8 cells. However, after adsorption, no extrachromosomal viral DNA in the cells was detected by the Southern blot method. In contrast, when MT-2 cells were cocultured with MOLT-4 # 8 cells, generation of extrachromosomal DNA was clearly observed. These data suggest that the cell-free HTLV-I may have difficulties in penetration, uncoating or reverse transcription. After cocultivation, MOLT-4 # 8 cells chronically infected with HTLV-I were cloned and analyzed. Only four provirus-positive cell lines were obtained. The transmission rate of the virus by cocultivation seemed to be low in our experimental system, although marked cell fusion was observed. Moreover, none of the cloned cell lines which harbored HTLV-I provirus expressed any viral protein. Inefficient integration and expression of the provirus might be hypothesized as compared with human immunodeficiency virus type 1 transmission.