Treatment of murine thymocytes, but not mature peripheral T cells, with the tumor promoter, phorbol 12-myristate 13-acetate (PMA), 3 results in a rapid disappearance of L3T4 molecules from the surface of thymocytes. The effect of PMA on L3T4 molecules persists in vitro for at least 72 hr. Down modulation of L3T4 molecules was PMA dose-dependent and temperature-dependent. L3T4 molecules on cortisone-resistant thymocytes were significantly less sensitive to the effect of PMA than were L3T4 molecules on cortisone-sensitive thymocytes. Down modulation of L3T4 molecules on thymocytes did not interfere with their capacity to respond to concanavalin A or activation signals delivered via their T cell receptors. The difference in the ability of thymocytes and peripheral T cells to respond to PMA cannot be explained by differences in the number of PMA receptors. Both thymocytes and peripheral T cells have PMA receptors in the range of 1 to 1.5 X 10(5) receptors/cell. However, there is a small difference in the affinity (Kd) of the receptors on thymocytes (Kd = 30 to 40 nM) and peripheral T cells (Kd = 10 to 15 nM). Immunofluorescent staining revealed that the down modulation of L3T4 molecules by PMA was a result of internalization of L3T4 molecules. After down modulation, L3T4 could be readily detected on the cytoplasm of thymocytes. These findings suggest that L3T4 molecules on thymocytes may be subject to different regulatory signals than L3T4 molecules on peripheral T cells.