We have recently demonstrated that the p38 and ERK1/2 MAP kinases play reciprocal roles in the control of CD1d-mediated antigen presentation. Although the use of specific inhibitors for these pathways clearly had an effect, the effects were not complete, leading to speculations that additional pathways were involved. Here, we show that inhibiting protein kinase C δ (PKCδ) substantially impairs antigen presentation by murine CD1d1 to NKT cells. This effect was accompanied by marked changes in the intracellular localization of CD1d. Expression of a dominant-negative mutant of PKCδ in CD1d+ cells resulted in nearly undetectable endogenous antigen presentation, substantially impaired CD1d recycling, a decrease in MAPK activation, and a decrease in the ability to present low (but not high) concentrations of α-galactosylceramide at the cell surface. These data strongly suggest that PKCδ is a critical regulator of CD1d-mediated antigen presentation and is involved in multiple steps of the process.