Increasing evidence suggests that reversible phosphorylation of histidine residues in proteins is important for signaling cascades in eukaryotic cells. Recently, the first eukaryotic protein histidine phosphatase (PHP) was identified. The beta(1)-subunit of heterotrimeric G proteins (G beta) undergoes phosphorylation on His(266) which is apparently involved in receptor-independent G protein activation. We studied whether phosphorylated G beta-subunits are substrates of PHP. Phosphorylated G beta gamma dimers of the retinal G protein transducin and G beta in membrane preparations of H10 cells (neonatal rat cardiomyocytes) were dephosphorylated by PHP. Overexpression of PHP in H10 cells showed that PHP and G beta also interfere within cells. In membranes of cells overexpressing PHP, the amount of phosphorylated G beta was largely reduced. Both our in vitro and cell studies indicate that phosphorylated G beta-subunits of heterotrimeric G proteins are substrates of PHP. Therefore, PHP might play a role in the regulation of signal transduction via heterotrimeric G proteins. (c) 2005 Elsevier Inc. All rights reserved.