Myosins are ATPase motor proteins that are activated by and traffic along actin filaments. This large protein family is divided into many classes with different functional properties and specializations for various roles, including membrane anchorage, longer range transport of cargo vesicles or cell signaling.Myosin class VI is unique due to its reversed directionality along actin filaments, moving towards the pointed end, in contrast to almost all other classes, which move towards the barbed end of F-actin. Whilst the directionality is well studied, other characteristics such as activation, cargo and lipid binding or dimerization are not fully understood. Using size exclusion chromatography, titration studies and gliding filament assays we investigated myosin VI backfolding, cargo binding and mechanical activity.Furthermore, we applied electron microscopy and single particle image processing to determine the structural properties of myosin VI in different ionic and nucleotide conditions. Two dimensional class averages based on various alignment and classification methods were made that allow for a detailed structural analysis including a comparison with crystal structures.