Regions of basic amino acids in proteins can promote membrane localization through electrostatic interactions with negatively charged membrane lipid head groups. Previous work showed that the heterotrimeric G protein subunit alpha(q) contains a polybasic region in its N terminus that contributes to plasma membrane localization. Here, the role of the N-terminal polybasic region of alpha(q) in signaling was addressed. For alpha(q) mutants, loss of plasma membrane localization correlated with loss of signaling function, as measured by the ability to couple activated G protein-coupled receptors (GPCRs) to stimulation of inositol phosphate production. However, recovery of plasma membrane localization of alpha(q) polybasic mutants by introduction of a site for myristoylation or by coexpression of beta gamma failed to recover signaling, suggesting a role for N-terminal basic amino acids of alpha(q) beyond simple plasma membrane localization. It is noteworthy that an alpha(q)4Q mutant, containing glutamine substitutions at arginines 27, 30, 31, and 34, was identified that failed to mediate signaling yet retained plasma membrane localization. Although alpha(q)4Q failed to couple activated receptors to inositol phosphate production, it was able to bind beta gamma, bind RGS4 in an activation-dependent manner, stimulate inositol phosphate production in a receptor-independent manner, and productively interact with a GPCR in isolated membranes. It is noteworthy that alpha(q)4Q showed a differing localization to plasma membrane nanodomains compared with wild-type alpha(q). Thus, basic amino acids in the N terminus of alpha(q) can affect its lateral segregation on plasma membranes, and changes in such lateral segregation may be responsible for the observed signaling defects of alpha(q)4Q.