It is widely accepted that the vesicle-transporter myosin-V moves processively along F-actin with large steps of similar to 36 nm using a hand-over-hand mechanism. A key question is how does the rear head of two-headed myosin-V search for the forward actin target in the forward direction. Scanning probe nanometry was used to resolve this underlying search process, which was made possible by attaching the head to a relatively large probe. One-headed myosin-V undergoes directional diffusion with similar to 5.5 nm substeps to develop an average displacement of similar to 20 nm,. which was independent of the neck length (2IQ and 6IQ motifs). Two-headed myosin-V showed several similar to 5.5 nm substeps within each processive similar to 36 nm step. These results suggest that the myosin-V head searches in the forward direction for the actin target using directional diffusion on the actin subunits according to a potential slope created along the actin helix. (c) 2006 Elsevier Inc. All rights reserved.