An immediate consequence of Ag-specific activation of T cells is phosphorylation of the gamma-subunit of the CD3 gamma-chain. There is good evidence that the kinase that mediates CD3 gamma-chain phosphorylation is protein kinase C (pkC). It has also been proposed that the interaction between pkC and CD3 gamma-chains controls the cell surface expression of the antigen receptor/CD3 Ag complex. In the present study we present data relevant to these two points. Thus we show that CD3 gamma-subunit phosphorylation can be triggered by the calcium ionophore ionomycin. However, as judged by several criteria, ionomycin does not stimulate cellular pkC. Accordingly, ionomycin must regulate phosphorylation of the CD3 Ag by a kinase distinct from pkC. The phosphorylation of CD3 Ag induced by ionomycin is not accompanied by a modulation of the cell surface expression of CD3 molecules which implies that CD3 gamma-chain phosphorylation is not a sufficient signal for the endocytosis of the CD3/Ag receptor complex.