Myosin V is a two-headed unconventional myosin that has an extended neck due to the presence of six light chain binding IQ-repeats. In cells, myosin V is thought to be a vesicle motor. Double-headed (HMM) and single-headed fragments (S1) of mouse myosin V, each tagged with the FLAG-epitope on their carboxyl-terminal end, were expressed in Sf9 cells along with calmodulin. Several lines of evidence suggest that myosin V is a processive motor which can move some distance along actin filaments without dissociation and that the two heads can bind simultaneously to the same actin filament. We have made mutants that increase or decrease the length of the neck region by increasing or decreasing the number of IQ residues. The wild-type myosin V has six IQ motifs. We find that the 4IQ, 6IQ and 8IQ myosin V molecules move processively in the TIRF assay and in the optical trap under most conditions tested and that the 2IQ mutant moves processively at low ATP concentrations. The step size of single events in the optical trap increases with the length of the neck as does the rate of in vitro motility. We have recently used a new microscopic technique termed FIONA (fluorescence imaging at one nanometer accuracy) to look at the stepping behavior of fluorescently-labeled myosin V molecules moving processively on actin. The data show that an N-terminally EGFP-tagged myosin V HMM, in which only one of the two heads are labeled, takes 74 nm steps, which is consistent with a hand-over-hand lever arm stepping model. FIONA studies with the neck length mutants show a linear relationship between the step size and the length of the neck. We have found that, under conditions in which the enzymatic activity of tissue purified myosin V is low (absence of calcium), the molecule is folded into a compact triangular shape. Upon activation of the ATPase activity by calcium or by increasing the ionic strength, the molecule opens up to adopt the familiar T- or Y-shaped structures.