It has been puzzled that in spite of its single-headed structure, myosin-IX shows the typical character of processive motor in multimolecule in vitro motility assay, because this cannot be explained by hand-over-hand mechanism of the two-headed processive myosins. Here, we show direct evidence of the processive movement of rnyosin-IX using two different single molecule techniques. Using optical trap nanometry, we found that rnyosin-IX takes several large (similar to 20 nm) steps before detaching from an actin filament. Furthermore, we directly visualized the single myosin-IX molecules moving on actin filaments for several hundred nanometers without dissociating from actin filament. Since myosin-IX processively moves without anchoring the neck domain, the result suggests that the neck tilting is not involved for the processive movement of myosin-IX. We propose that the rnyosin-IX head moves processively along all actin filament like an inchworm via a unique long and positively charged insertion in the loop 2 region of the head. (c) 2006 Elsevier Inc. All rights reserved.