Angiotensin I–converting enzyme (ACE) inhibitors are thought to lower blood pressure in hypertensive patients, mainly by decreasing angiotensin II (Ang II) formation. Chymase, a human mast cell protease, has recently been proposed to play a role in blood pressure regulation because of its Ang II–forming activity. Here we show that the predominant chymase mRNA species in the mouse aorta are those for types 4 and 5 isoforms, and that both are efficient Ang II–forming enzymes. Evaluation of ACE-dependent and ACE-independent Ang II–forming pathways in mast cell–deficient (Kit^w/Kit^w-v) mice and their mast cell–sufficient littermate (MC^+/+) controls revealed that, in contrast to the latter, Kit^w/Kit^w-v mice fail to express chymase mRNAs in the vasculature and have almost no ACE-independent Ang II–forming activity in either isolated blood vessels or homogenates. Moreover, in MC^+/+ but not in Kit^w/Kit^w-v mice, a contribution of ACE-independent Ang II generation to blood pressure regulation was evident by a 1.6-fold greater maximal reduction in mean arterial pressure with acute ACE inhibition plus AT₁ receptor blockade than with ACE inhibition alone. Thus, mast cells are the source of the vascular ACE-independent pathway, and the antihypertensive benefit of combining ACE inhibitor therapy with AT₁ receptor antagonist therapy is most likely due to negation of chymase-catalyzed Ang II generation.