The sphingolipid ceramide plays a role in receptor clustering in the plasma membrane. Upon bacterial encounter, dendritic cells (DCs) initiate a bacteria-specific downstream signalling event. We hypothesized that conversion of sphingomyelin to ceramide by acid sphingomyelinase (SMase) is a key event in endocytosis of gram-positive Lactobacillus acidophilus and the subsequent induction of IFN-β in DCs. Conversely, endocytosis of the gram-negative Escherichia coli, which is not a potent IFN-β inducer would not be dependent on ceramide formation. SMase or an inhibitor of acid SMase and ceramidase, chlorpromazine (CPZ), was added to murine bone marrow (bm)DCs prior to stimulation with either of the bacteria. Simultaneous endocytosis of fluorescent-labelled bacteria and FITCdextran measured by flow cytometry provided a method to distinguish between phagocytosis, constitutive macrocytosis, and induced macropinocytosis in the bmDCs. Addition of SMase increased the phagocytosis of L. acidophilus and L. acidophilus-induced IL-12/IFN-β but showed no effect on the uptake of E. coli nor on E.coli induced IL-12/IFN-β production. Also, SMase did not affect Pam3CSK4-induced macropinocytosis of FITC-dextran. Inhibition of both acid SMase and ceramidase by CPZ increased constitutive macropinocytosis of dextran and slightly increased L.acidophilus induced Il12/Ifn-β expression and E.coli induced Ifnβ expression. Our results confirm a role for ceramide in the L.acidophilus induced IL-12/IFN-β production but also enhancement of constitutive micropinocytosis by inhibiting sphingomyelin conversion may lead to enhanced IFN-β induction. Our data suggests that manipulation of the membrane sphingolipids provides a tool for manipulating the cytokine profiles in DCs, e.g. in vaccine development.