Prostaglandin E2 (PGE2) is the predominant eicosanoid product released by macrophages at the site of inflammation. Binding of PGE2 to its cognate 7 transmembrane-spanning G protein-coupled receptors (GPCRs) activates signaling pathways, leading to the synthesis of the Fos transcription factor. Because the Ste20 serine/threonine protein kinase (S/TPK) is a critical signal transducer for the G protein-coupled pheromone receptor in Saccharomyces cerevisiae, we postulated that the PGE2 GPCRs may activate one of the Ste20 mammalian orthologs. We demonstrate here that the catalytic activity of a hematopoletic cell-restricted, Ste20-related S/TPK, HPK1, is positively regulated by exposure to physiological concentrations of PGE2. Furthermore, ectopic expression studies implicated HPK1 as a negative regulator of PGE2-induced transcription of the fos gene. Our data suggest that PGE2-induced activation of HPK1 may represent a novel negative regulatory pathway capable of modulating PGE2-mediated gene transcription.