An increased migratory phenotype exists in lung fibroblasts derived from patients with fibroproliferative lung disease. Prostaglandin E(2) (PGE(2)) suppresses fibroblast migration, but the receptor(s) and mechanism(s) mediating this action are unknown. Our data confirm that treatment of human lung fibroblasts with PGE(2) inhibits migration. Similar effects of butaprost, an E-prostanoid (EP) 2 receptor-specific ligand, implicate the EP2 receptor in migration-inhibitory signaling. Further, migration in fibroblasts deficient for the EP2 receptor cannot be inhibited by PGE(2) or butaprost, confirming the central role of EP2 in mediating these effects. Our previous data suggested that phosphatase and tensin homolog on chromosome ten (PTEN), a phosphatase that opposes the actions of phosphatidylinositol-3-kinase (PI3K), may be important in regulating lung fibroblast motility. We now report that both PGE(2) and butaprost increase PTEN phosphatase activity, without a concomitant increase in PTEN protein levels. This contributes to EP2-mediated migration inhibition, because migration in PTEN-null fibroblasts is similarly unaffected by EP2 receptor signaling. Increased PTEN activity in response to EP2 stimulation is associated with decreased tyrosine phosphorylation on PTEN, a mechanism known to regulate enzyme activity. Collectively, these data describe the novel mechanistic finding that PGE(2), via the EP2 receptor, decreases tyrosine phosphorylation on PTEN, resulting in increased PTEN enzyme activity and inhibition of fibroblast migration.