1. The [Ca2+] sensitivity of myosin light chain phosphorylation in vascular smooth muscle is dependent on the form of stimulation. Contractile agonist stimulation, when compared to high-KCl depolarization, is associated with an increase in [Ca2+] sensitivity of phosphorylation. I evaluated potential mechanisms for this stimulus-specific response by measuring aequorin-estimated myoplasmic [Ca2+], myosin phosphorylation, and isometric stress in swine carotid media. 2. The relative [Ca2+] sensitivity of phosphorylation depended on the type of stimulus (ranked high to low sensitivity): contractile agonists (histamine, phenylephrine) = endothelin (sustained contraction) = combination of histamine and NaF greater than NaF alone = endothelin (initial contraction) = combination of histamine and depolarization = combination of NaF and depolarization greater than depolarization = Bay K 8644 = combination of depolarization and low-dose phorbol diester. 3. Activation of L-type Ca2+ channels with Bay K 8644 induced a [Ca2+] sensitivity of phosphorylation similar to depolarization, suggesting that any other effects of high KCl (such as cellular swelling) were not responsible for the low [Ca2+] sensitivity of phosphorylation. 4. The addition of either histamine or NaF (an activator of G proteins) to depolarized tissues produced similar increases in the [Ca2+] sensitivity of phosphorylation, suggesting that NaF (possibly by activation of a G protein) can mimic contractile agonist-induced increases in the [Ca2+] sensitivity of phosphorylation. 5. Phorbol dibutyrate enhanced the contractile effect of depolarization, and this enhancement was primarily caused by increases in [Ca2+] rather than an alteration in the [Ca2+] sensitivity of phosphorylation. 6. These data suggest that the [Ca2+] sensitivity of phosphorylation in smooth muscle may be regulated by agonists (possible by G protein activation); however, the role of protein kinase C activation or depolarization induced Ca2+ compartmentalization requires further study.