G-protein-coupled receptors (GPCRs) typically activate c-Jun N-terminal kinase (JNK) through the G protein beta gamma subunit (G beta gamma), in a manner dependent on Rho family small GTPases, in mammalian cells. Here we show that JNK activation by the prototypic Gp-coupled alpha 1B-adrenergic receptor is mediated by the a subunit of Gq (G alphaq), not by G beta gamma, using a transient transfection system in human embryonic kidney cells. JNK activation by the alpha 1B-adrenergic receptor/G alphaq was selectively mediated by mitogen-activated protein kinase kinase 4 (MKK4), but not MKK7. Also, MKK4 activation by the alpha 1B-adrenergic receptor/G alphaq required c-Src and Rho family small GTPases. Furthermore, activation of the alpha 1B-adrenergic receptor stimulated JNK activity through Src family tyrosine kinases and Rho family small GTPases in hamster smooth muscle cells that natively express the alpha 1B-adrenergic receptor. Together, these results suggest that the alpha 1B-adrenergic receptor/G alphaq may up-regulate JNK activity through a MKK4 pathway dependent on c-Src and Rho family small GTPases in mammalian cells.