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Diverse extracellular signals induce plasma membrane translocation of sphingosine kinase-1 (SphK1), thereby enabling inside-out signaling of sphingosine-1-phosphate. We have shown before that G q -coupled receptors and constitutively active Gα q/11 specifically induced a rapid and long-lasting SphK1 translocation, independently of canonical G q /phospholipase C (PLC) signaling. Here, we further characterized G q/11 regulation of SphK1. SphK1 translocation by the M 3 receptor in HEK-293 cells was delayed by expression of catalytically inactive G-protein-coupled receptor kinase-2, p63Rho guanine nucleotide exchange factor (p63RhoGEF), and catalytically inactive PLCβ 3 , but accelerated by wild-type PLCβ 3 and the PLCδ PH domain. Both wild-type SphK1 and catalytically inactive SphK1-G82D reduced M 3 receptor-stimulated inositol phosphate production, suggesting competition at Gα q . Embryonic fibroblasts from Gα q/11 double-deficient mice were used to show that amino acids W263 and T257 of Gα q , which interact directly with PLCβ 3 and p63RhoGEF, were important for bradykinin B 2 receptor-induced SphK1 translocation. Finally, an AIXXPL motif was identified in vertebrate SphK1 (positions 100-105 in human SphK1a), which resembles the Gα q binding motif, ALXXPI, in PLCβ and p63RhoGEF. After M 3 receptor stimulation, SphK1-A100E-I101E and SphK1-P104A-L105A translocated in only 25% and 56% of cells, respectively, and translocation efficiency was significantly reduced. The data suggest that both the AIXXPL motif and currently unknown consequences of PLCβ/PLCδ(PH) expression are important for regulation of SphK1 by G q/11 .