Heterotrimeric G proteins typically localize at the cytoplasmic face of the plasma membrane where they interact with heptahelical receptors. For G protein alpha subunits, multiple membrane targeting signals, including myristoylation, palmitoylation, and interaction with betagamma subunits, facilitate membrane localization. Here we show that an additional membrane targeting signal, an N-terminal polybasic region, plays a key role in plasma membrane localization of non-myristoylated alpha subunits. Mutations of N-terminal basic residues in alpha(s) and alpha(q) caused defects in plasma membrane localization, as assessed through immunofluorescence microscopy and biochemical fractionations. In alpha(s), mutation of four basic residues to glutamine was sufficient to cause a defect, whereas in alpha(q) a defect in membrane localization was not observed unless nine basic residues were mutated to glutamine or if three basic residues were mutated to glutamic acid. betagamma co-expression only partially rescued the membrane localization defects; thus, the polybasic region is also important in the context of the heterotrimer. Introduction of a site for myristoylation into the polybasic mutants of alpha(s) and alpha(q) recovered strong plasma membrane localization, indicating that myristoylation and polybasic motifs may have complementary roles as membrane targeting signals. Loss of plasma membrane localization coincided with defects in palmitoylation. The polybasic mutants of alpha(s) and alpha(q) were still capable of assuming activated conformations and stimulating second messenger production, as demonstrated through GST-RGS4 interaction assays, cAMP assays, and inositol phosphate assays. Electrostatic interactions with membrane lipids have been found to be important in plasma membrane targeting of many proteins, and these results provide evidence that basic residues play a role in localization of G protein alpha subunits.