We investigated the role of myosin light chain kinase (MLCK) phosphorylation in regulating the sensitivity of vascular smooth muscle myosin light chain (MLC) phosphorylation to intracellular Ca2+ concentration ([Ca2+]i). 32PO4-loaded swine carotid arteries were stimulated with histamine or high K+, MLCK was isolated, and the relative phosphorylation of tryptic peptides was measured. In nonlabeled tissues, we measured [Ca2+]i with aequorin, MLCK activity ratio, MLC phosphorylation, and force. A comparison of MLCK phosphorylation on peptide A (mol P in site A/mol MLCK) and MLCK activity ratio showed an inverse relation, suggesting that MLCK site A phosphorylation can regulate the Ca2+ sensitivity of MLCK. MLCK site A phosphorylation and MLCK activity ratio depended on [Ca2+]i. Histamine stimulation yielded greater MLC phosphorylation than high K+ stimulation over a range of [Ca2+]i; however, there were no apparent stimulus-dependent differences in MLCK phosphorylation, suggesting that stimulus-dependent differences in the Ca2+ sensitivity of MLC phosphorylation are not based on differences in MLCK phosphorylation. We also determined whether MLCK phosphorylation was involved in adenosine 3',5'-cyclic monophosphate-mediated relaxation. In histamine-contracted tissues, forskolin decreased [Ca2+]i, MLC phosphorylation, and force. MLCK phosphorylation decreased to an extent consistent with the decrease in [Ca2+]i. In KCl-stimulated tissues, forskolin did not alter [Ca2+]i or increase MLCK phosphorylation but forskolin did decrease MLC phosphorylation. Thus, in swine carotid artery, MLCK phosphorylation appears to be regulated exclusively by Ca2+ and plays little role in stimulus-dependent differences in Ca2+ sensitivity of MLC phosphorylation or in mediating forskolin-induced relaxation.