Phosphoinositides are important signaling molecules that govern a large number of cellular processes such as proliferation, differentiation, membrane remodeling, and survival. Here we introduce a fully synthetic membrane-permeant derivative of a novel, easily accessible, and very potent phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] mimic: phosphatidylinositol 3,4,5,6-tetrakisphosphate [PtdIns(3,4,5,6)P(4)]. The membrane-permeant PtdIns(3,4,5,6)P(4) derivative activated pathways downstream of phosphatidylinositol 3-kinase (PI3K), including protein kinase B, p70S6K, mitogen-activated protein kinase, and protein kinase C, more potently than similar membrane-permeant PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) derivatives in the absence of receptor stimulation. In addition, we demonstrate that treatment of PC12 cells with the membrane-permeant PtdIns(3,4)P(2), PtdIns(3,4,5)P(3), and PtdIns(3,4,5,6)P(4) derivatives increases the number of neurites per cell in the presence of NGF. This work establishes membrane-permeant phosphoinositides as powerful tools to study PI3K signaling and directly demonstrates that 3-phosphorylated phosphoinositides are instrumental for neurite initiation.