PLC-ε was identified recently as a phosphoinositide-hydrolyzing phospholipase C (PLC) containing catalytic domains (X, Y, and C2) common to all PLC isozymes as well as unique CDC25- and Ras-associating domains. Novel regulation of this PLC isozyme by the Ras oncoprotein and α-subunits (Gα12) of heterotrimeric G proteins was illustrated. Sequence analyses of PLC-ε revealed previously unrecognized PH and EF-hand domains in the amino terminus. The known interaction of Gβγ subunits with the PH domains of other proteins led us to examine the capacity of Gβγ to activate PLC-ε. Co-expression of Gβ 1γ2 with PLC-ε in COS-7 cells resulted in marked stimulation of phospholipase C activity. Gβ2 and Gβ4 in combination with Gγ1, Gγ 2, Gγ3, or Gγ13 also activated PLC-ε to levels similar to those observed with Gβ 1-containing dimers of these Gγ-subunits. Gβ3 in combination with the same Gγ-subunits was less active, and Gβ 5-containing dimers were essentially inactive. Gβγ -promoted activation of PLC-ε was blocked by cotransfection with either of two Gβγ-interacting proteins, Gαi1 or the carboxyl terminus of G protein receptor kinase 2. Pharmacological inhibition of PI3-kinase-γ had no effect on Gβ1γ 2-promoted activation of PLC-ε. Similarly, activation of Ras in the action of Gβγ is unlikely, because a mutation in the second RA domain of PLC-ε that blocks Ras activation of PLC failed to alter the stimulatory activity of Gβ1γ2. Taken together, these results reveal the presence of additional functional domains in PLC-ε and add a new level of complexity in the regulation of this novel enzyme by heterotrimeric G proteins.