HTLV-I has recently been shown to be a direct activator of resting human peripheral T cells. In order to determine the susceptibility of T-cell precursors to HTLV-I mitogenic activity we have exposed human thymic T cells to uv-inactivated HTLV-I. Unlike mature T cells, thymocytes were not directly susceptible to HTLV-I-induced activation although agglutination of cells did occur after exposure to HTLV-I alone. However, in the presence of another stimulus, phyto-hemagglutinin or anti-CD3 monoclonal antibodies and accessory cells, thymocytes proliferated when exposed to HTLV-I. Concanavalin A did not induce HTLV-I comitogenic activity. HTLV-I-induced thymocyte proliferation was enhanced by autologous or heterologous accessory cells. This proliferation was shown to be mediated by the interleukin-2/interleukin-2 receptor pathway. Simultaneous stimulation by HTLV-I and nonmitogenic doses of phytohemagglutinin were required both for the production of interleukin-2 and for the expression of the interleukin-2 receptor. These data demonstrated functional differences between peripheral T cells and thymocytes.