3-Phosphoinositide-dependent kinase 1 (PDK1) has previously been shown to phosphorylate the activation loop of several AGC kinase family members. In this study, we show that p21-activated kinase 1, the activity of which is regulated by the GTP-bound form of Cdc42 and Rac and by sphingosine, is phosphorylated by PDK1. Phosphorylation of p21-activated kinase 1 by PDK1 occurred only in the presence of sphingosine, which increased PDK1 autophosphorylation 25-fold. Sphingosine increased PDK1 autophosphorylation in a concentration-dependent manner and significantly increased phosphate incorporation into known PDK1 substrates. Studies on the lipid requirement for PDK1 activation found that both sphingosine isoforms and stearylamine also increased PDK1 autophosphorylation. However, C(10)-sphingosine, octylamine, and stearic acid were unable to increase PDK1 autophosphorylation, indicating that both a positive charge and a lipid tail containing at least a C(10)-carbon backbone were required for PDK1 activation. Three PDK1 autophosphorylation sites were identified after stimulation with sphingosine in a serine-rich region located between the kinase domain and the pleckstrin homology domain using two-dimensional phosphopeptide maps and matrix assisted laser desorption/ionization mass spectroscopy. Increased phosphorylation of endogenous Akt at threonine 308 was observed in COS-7 cells expressing wild type PDK1, but not catalytically inactive PDK1, when cellular sphingosine levels were elevated by treatment with sphingomyelinase. Sphingosine thus appears to be a true PDK1 activator.