Tsk/Itk and Btk are members of the pleckstrin-homology (PH) domain-containing tyrosine kinase family. The PH domain has been demonstrated to be able to interact with beta gamma subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) (G beta gamma) and phospholipids. Using cotransfection assays, we show here that the kinase activities of Tsk and Btk are stimulated by certain G beta gamma subunits. Furthermore, using an in vitro reconstitution assay with purified bovine brain G beta gamma subunits and the immunoprecipitated Tsk, we find that Tsk kinase activity is increased by G beta gamma subunits and another membrane factor(s). These results indicate that this family of tyrosine kinases could be an effector of heterotrimeric G proteins.