Pleckstrin homology (PH) domains are found in numerous proteins important in signal transduction and cytoskeletal function. Several PH domains are now known to contain a binding site for the beta gamma subunits of trimeric G-proteins (G beta gamma), a finding which naturally raises the question of where on the G beta gamma complex these PH domains bind. Here we demonstrate binding of the PH domains of beta-adrenergic receptor kinase and beta-spectrin to the G beta subunit and not the G gamma subunit in a nitrocellulose gel replica assay. Furthermore, the C-terminal tryptic fragment of G beta containing only 5 WD40/beta-transducin (WD40) repeats also binds these two PH domains. Finally, constructs containing only WD40 repeats of G beta were shown to bind to beta-ARK and beta-spectrin PH domains in solution. These findings suggest that WD40 repeats of G beta are ligands for PH domains and have interesting implications for other proteins containing WD40 sequences.