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Peripheral blood mononuclear cells (PBMC) from three adult male squirrel monkeys (Saı̈miri sciureus) were transformed by human T-cell leukemia/lymphoma virus type I (HTLV-I) by cocultivation with lethally irradiated human MT-2 cells. Three permanent monkey T-cell lines producing HTLV-I were obtained and characterized. Six weeks after inoculation seroconversion was observed in three of three monkeys inoculated with autologous transformed T cells and in two of three monkeys receiving homologous cells. Proviral DNA was detected in their PBMC at various times after inoculation, with the highest proviral load and antibody titers being found in monkeys infected with homologous cells. Monkeys inoculated with heterologous MT-2 cells did not seroconvert, and HTLV-I provirus was detected only transiently in their PBMC. To determine whetherin vitroandin vivoHTLV-I infection of squirrel monkey cells led to a selection of monkey-adapted viral mutants, comparative sequencing of the proviral gp21envbetweenex vivomonkey HTLV-I-infected PBMC, the inoculum, and MT-2 cells was done and no significant differences were detected. The squirrel monkey, which is naturally free of simian T-cell leukemia/lymphoma virus, thus appears to be a suitable model for evaluating HTLV-I candidate vaccines and for studying the pathogenesis of HTLV-I.