The D2 dopamine receptor (D2R) was examined for its ability to mediate nuclear factor-κB (NF-κB) activation through G proteins. Stimulation of D2R-transfected HeLa cells with its agonist quinpirole induced the expression of a NF-κB luciferase reporter and formation of NF-κB-DNA complex. This response was blocked by pertussis toxin, and by the Gβγ scavengers transducin and β-adrenergic receptor kinase 1 carboxyl-terminal fragment. Unlike G-coupled chemoattractant receptors, D2R activated NF-κB without an increase in phospholipase C-β activity, and the response was only slightly affected by the phosphoinositide 3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). In contrast, treatment with genistein and 4-amino-1-tert-butyl-3-(p-methylphenyl)pyrazolo[3,4-d] pyrimidine abolished the induced NF-κB activation, suggesting involvement of protein tyrosine kinases. Activation of D2R led to phosphorylation of c-Src at Tyr-418 and expression of a kinase-deficient c-Src inhibited D2R-mediated NF-κB activation. The D2R-mediated NF-κB activation was not dependent on epidermal growth factor (EGF) receptor transactivation since 4-(3′-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), an EGF receptor-selective tyrphostin used at 1 μM, blocked EGF-induced NF-κB activation but not the quinpirole-induced response. In addition, the D2R-mediated NF-κB activation was enhanced by over-expression of β-arrestin 1. These results suggest that D2R-mediated NF-κB activation requires Gβγ and c-Src, and possibly involves β-arrestin 1.