There are three myosin V genes in mammals, termed Va, Vb and Vc. Recent studies provide strong evidence that single class V myosin molecules transport vesicles and organelles processively along F-actin, taking several 36-nm steps, hand over hand, for each diffusional encounter. We demonstrated that the ATPase activity of myosin required calcium for maximal activity and showed that, in the absence of calcium, myosin V adopted a folded, inactive structure. We have used negative staining and cryo-electron microscopy to examine the structure of myosin V that is walking on actin. These images give clear pictures of myosin V molecules with both heads attached to actin and will allow us to make observations about lever arm position and stiffness. We are examining the structure of actin-bound myosin V mutants in which the spacing between IQ motifs were altered and will complement these studies with in vitro motility assays and optical trapping. We also examined the structure of mutant myosin V molecules in which the neck region was altered by changing the number of IQ motilfs. These studies demonstrate that the neck of myosin V has evolved to allow it to take 36 nm strides along actin which matches the helical repeat. We have mutated the switch-1 region of myosin V (S217A) and found that this mutant alters the duty ratio and effects processivity of the motor. Switch-2 mutants affect the speed of translocation and the ADP release rate. Optical trapping studies show that under certain levels of strain, the powerstroke of myosin V attached to actin can be reversed which may aid in maintaining myosin attachment during stall. We have begun to study the mechanical properties of myosin Vc and the regulatory properties of myosin Vb.