Phospholipase Cepsilon (PLCepsilon) is a novel PLC that has a CDC25 guanine nucleotide exchange factor (GEF) domain and two Ras association (RA) domains of which the second (RA2) is critical for Ras activation of the enzyme. In the present studies, we examined hormonal stimulation to elucidate receptor-mediated pathways that functionally regulate PLCepsilon. We demonstrate that epidermal growth factor (EGF), a receptor tyrosine kinase (RTK) agonist, and lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P), and thrombin, G protein-coupled receptor agonists, stimulate PLCepsilon overexpressed in COS-7 cells. EGF stimulated PLCepsilon in an RA2-dependent manner through Ras and Rap. In contrast, LPA, S1P and thrombin stimulated PLCepsilon by both RA2-independent and dependent mechanisms. To determine the G proteins that mediate the effects of these GPCR agonists, we coexpressed constitutively active G proteins with PLCepsilon and found that Galpha12, Galpha13, Rho, Rac and Ral stimulate PLCepsilon in an RA2-independent manner; whereas, TC21, Rap1A, Rap2A and Rap2B stimulate in an RA2-dependent manner similar to H-Ras. Of these G proteins, we show that Galpha12/13 and Rap partly mediate the effects of LPA, S1P, and thrombin to stimulate PLCepsilon. In addition, the stimulation by LPA and S1P is also partly sensitive to pertussis toxin. These studies demonstrate diverse hormonal regulation of PLCepsilon by distinct and overlapping pathways.