SH-PTP1 is a protein-tyrosine phosphatase preferentially expressed in hematopoietic cells and bearing two SH2 (src homology-2) domains. In the human megakaryocytic cell line Dami, lysophosphatidic acid (LPA) promoted a rapid increase in SH-PTP1 phosphorylation on both serine and tyrosine residues. Only tyrosine phosphorylation was significantly inhibited by pertussis toxin and by the protein kinase C inhibitor GF109203X. Moreover, SH-PTP1 was phosphorylated upon challenge with other agonists acting via G-protein-coupled receptors such as alpha-thrombin, epinephrine, and ADP, whereas the closely related protein-tyrosine phosphatase SH-PTP2 failed to share such a regulation in Dami cells. We developed an in vitro assay that reproduced LPA-dependent phosphorylation of SH-PTP1 in a cell-free system. The fusion protein glutathione S-transferase-beta-adrenergic receptor kinase 1-(495-689) or the transducin subunit Galphat-GDP, which act as specific antagonists of Gbetagamma, inhibited SH-PTP1 phosphorylation. Moreover, purified transducin Gbetagamma subunits mimicked the effect of LPA. Finally, stable expression of beta-adrenergic receptor kinase 1-(495-689) in Dami cells resulted in the inhibition of SH-PTP1 as a specific target of protein kinases linked to G-protein-coupled receptors via Gbetagamma subunits.