We have succeeded in infecting human thymic lymphocytes with both the HIV-IIIB laboratory strain of HIV-1 as well as with a clinical isolate of this virus. Thymic lymphocytes were at least as susceptible to infection by HIV-1 as were cord blood lymphocytes, but appeared to display somewhat greater resistance to the cytopathic effects of the virus. As measured variously by each of indirect immunofluorescence for detection of viral p17, antigen capture assay for the presence of viral p24 in culture fluids, and levels of viral reverse transcriptase activity in culture fluids, infection of thymic lymphocytes could be detected as early as 2 days after infection by HIV-1, and persisted through at least 14 days of tissue culture maintenance. These findings suggest that thymic lymphocytes may be susceptible to infection by HIV-1 in vivo, and may also be relevant to our understanding of HIV-1-induced pathogenesis, particularly in pediatric populations.