Little is known of the mechanisms leading to mitogen-activated protein kinase (MAPK) activation via Gq-coupled receptors. We there- fore examined the pathways by which angiotensin II (Ang II) activates Raf-1 kinase, an upstream intermediate in the pathway to MAPK, via the Gq-coupled AT1 angiotensin receptor in bovine adrenal glomeru- losa (BAG) cells. Ang II caused a rapid and transient activation of Raf-1 that reached a peak at 5-10 min. Ang II was a potent stimulus of Raf-1 activation with an ED50 of 10 pM and a maximal response at 1n M, although higher Ang II concentrations elicited a submaximal response. Ang II-stimulated Raf-1 activity was unaffected by down- regulation of protein kinase C and intracellular Ca21 chelation (using BAPTA) but was partially inhibited by pertussis toxin, and was abol- ished by manumycin A. Removal of extracellular Ca21 (by EGTA) or blockade of L type Ca21 channels (by nifedipine), as well as inhibition of MEK-1 kinase (by PD98059), enhanced Raf-1 activity, whereas wortmannin (100 nM) inhibited approximately one half of Ang II- stimulated Raf-1 activity. Hence, Raf-1 kinase activation by Ang II in BAG cells is dependent on Ras, is mediated in part via Gi and phos- phatidylinositol 3-kinase, and is negatively regulated via Ca21 influx and a downstream signaling element(s). (Endocrinology 140: 1385- 1391, 1999)