Myosin V isolated from chick brain (BM V) is a multimeric protein of about 640 kDa consisting of two intertwined heavy chains of 212 kDa and multiple light chains of 10 to 20 kDa. A distinctive feature of the heavy chain is an extended neck region with six consensus IQ sites for the binding of calmodulin (CaM) and myosin light chains. The actin-activated MgATPase has been shown to require ≥1 μM Ca2+for full activity, and evidence points to a myosin-linked regulatory system where the CaM light chains participate as modulators for the Ca2+signal. Still, the precise mechanism of Ca2+regulation remains unknown. In the present study we have used the intrinsic tryptophan fluorescence of native BM V to monitor conformational changes of BM V induced by Ca2+, and we relate these changes to CaM dissociation from the BM V molecule. The fluorescence intensity decreases ∼17% upon addition of sub-micromolar concentrations of Ca2+(K0.5= 0.038 μM). This decrease in fluorescence, which is dominated by a conformational change in the heavy chain, can be reversed by addition of 1,2-di(2-aminoethoxy)ethane-N,N,N′,N′tetraacetic acid (EGTA) followed by an excess of CaM, but not by addition of EGTA alone. Gel filtration of native BM V using HPLC shows that CaM is partially dissociated from the heavy chain in EGTA and dissociates further upon addition of sub-micromolar concentrations of Ca2+. These observations suggest that the affinity of CaM for at least one of the IQ sites on the BM V heavy chain decreases with Ca2+and that the Ca2+concentration required for this effect is lower than that needed to activate acto-BM V. Using a cosedimentation assay in the presence of actin, we also observe partial dissociation of CaM when Ca2+is absent, but now the addition of Ca2+has a biphasic effect: sub-micromolar Ca2+concentrations lead to reassociation of CaM with the heavy chain, followed by dissociation when Ca2+exceeds 5-10 μM. Thus, the binding of CaM to BM V is affected by both actin and Ca2+.