The function of mast cells is modified by the phosphoinositol-3 (PI3)-kinase pathway. The kinase signals partially through the phosphoinositide-dependent kinase PDK1, which on the one hand activates the serum- and glucocorticoid- inducible kinase SGK1 and on the other hand activates protein kinase PKCδ. SGK1 participates in the stimulation of Ca(2+) entry and degranulation, PKCδ inhibits degranulation. The present experiments explored the role of PDK1 in mast cell function. As mice completely lacking PDK1 are not viable, experiments have been performed in mast cells isolated from bone marrow (BMMCs) of PDK1 hypomorphic mice (pdk1(hm)) and their wild-type littermates (pdk1(wt)). Antigen stimulation via the FceRI receptor was followed by Ca(2+) entry leading to increase of cytosolic Ca(2+) activity in pdk1(wt) BMMCs, an effect significantly blunted in pdk1(hm) BMMCs. In contrast, Ca(2+) release from intracellular stores was not different between BMMCs of the two genotypes. The currents through Ca(2+)-activated K(+) channels following antigen exposure were again significantly larger in pdk1(wt) than in pdk1(hm) cells. The Ca(2+) ionophore ionomycin (1 μM) increased the K(+) channel conductance to similar values in both genotypes. β-hexosaminidase and histamine release were similar in pdk1(wt) BMMCs and pdk1(hm) BMMCs. PKCδ inhibitor rottlerin increased β-hexosaminidase release in pdk1(wt) BMMCs but not in pdk1(hm) BMMCs. Phosphorylation of PKCδ and of the SGK1 target NDRG1, was stimulated by the antigen in pdk1(wt) but not in pdk1(hm) cells. The observations reveal a role for PDK1 in the regulation of Ca(2+) entry into and degranulation of murine mast cells.