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We describe an approach that allows us to form a micro in vitro motility assay with as little myosin as can be retrieved from a short ( ∼ 10 mm) segment of a single skinned skeletal muscle fiber (diameter some 100 μm). Myosin is directly extracted from the single fiber segment by a high ionic strength solution in the presence of MgATP, and the extracted myosin is immediately applied to a miniaturized flow cell that has been pretreated with BSA. The observed sliding velocities of fluorescently labeled F-actin are essentially identical with those reported in the literature. Since at the single fiber level most muscle fibers contain only a single myosin heavy chain isoform this approach allows us to determine without additional purification steps, the sliding velocity driven by myosins with different heavy chain isoforms. In addition, this approach can be used to directly correlate under identical experimental conditions unloaded shortening velocity measured in segments of skinned muscle fibers with the in vitro sliding velocity of fluorescently labeled F-actin by extraction of myosin from the same skinned fibers. Such direct correlation was performed with different myosin heavy chain isoforms as well as at different temperatures and ionic strengths. Under all conditions studied, unloaded shortening velocity was 4- to 8-fold faster than sliding velocity in the motility assay even at high temperature (22∘ C) and ionic strengths >50 mM. This suggests that sliding velocity in the motility assay is limited by additional factors beyond those thought to limit velocity of unloaded shortening in muscle fibers. One such factor might be unspecific ionic interactions between F-actin and the substrate in the motility assay resulting in somewhat higher sensitivity for ionic strength of sliding velocity in the motility assay. This might become of special relevance when using in vitro sliding velocity in assessing functional consequences of mutations involving charged residues of actin or myosin.