Inter-molecular interactions between two smooth muscle myosin heads have been observed using chemical cross linking and visualized by electron microscopy. In order to probe the nature of the phosphorylation-dependent head-head interactions, we performed Hydrogen-Deuterium exchange electrospray ionization mass spectrometry (HDX-MS) on phosphorylated and unphosphorylated smooth muscle heavy mero myosin (HMM) from chicken gizzard. HDX rates are determined by solvent accesibility and the strength of hydrogen bonding and thus are a good fingerprint of intra- and intermolecular structural changes. The observed decrease in deuterium uptake upon phosphorylation in the coiled-coil and the hinge region of the heavy chain (residues 864-877), is consistent with the unraveling of the myosin dimer at the hinge. Unexpectedly, we have observed phosphorylation dependent changes in the regulatory light chain (RLC 58-69), which are not implicated in the RLC-RLC or RLC-heavy chain interface. No changes were observed for N-terminal region of RLC that was postulated to undergo major secondary structure changes, or in the heavy chain actin binding site (residues 397-437), or any of the residues that form the head-head interface in the EM model of Wendt & Taylor. This latter finding indicates that the interaction between the heads is probably transient in solution, or in the unlikely case, interacting heads constitute a very small fraction of total protein.