Phorbol esters are known to induce a loss of CD4 from the surface of mouse and human T cells, presumably through activation of protein kinase C. Here we describe additional, calcium-dependent processes that remove CD4 and CD8 from the surface of T cells and thymocytes, and that differ from the protein kinase C-mediated effect in that they require the expression of new gene products. Whereas PMA causes the disappearance of almost all CD4 from the surface of mouse CD4+8+ thymocytes, it induces only a partial (approximately 60%) and transient (10 to 12 h) loss of CD4 from the surface of mouse peripheral T cells, with no effect on CD8 expression. When T cells are exposed to a combination of PMA and the calcium ionophore, ionomycin (Cal), surface CD4 virtually disappears for a period of at least 24 h, and CD8 expression is also diminished. This additional, calcium-dependent effect, on both CD4 and CD8 expression, is abrogated by either cycloheximide or actinomycin D, and so depends on new RNA and protein synthesis. There appear, therefore, to be two distinct mechanisms for the removal of CD4 and CD8 from mouse peripheral T cells: one induced by PMA alone, the second by the combination of PMA and Cal. The second mechanism, but not the first, depends on the expression of new gene products. In contrast to mouse peripheral T cells, mouse thymocytes and human peripheral blood T cells respond to PMA alone with virtually a complete loss of surface CD4, and partial loss of CD8 expression. The addition of Cal has no synergistic effect on CD4 expression in either of these populations, but augments the loss of CD8 in mouse thymocytes.