Publisher Summary This chapter discusses the biophysics of dynein protein. It provides an understanding of molecular motors, whose function is to physically exert force and move cargos of a variety of types from one place to another inside cells, from an enzymatic point of view and from a biophysical one. It summarizes what is known about how single-molecule properties relate to ensemble function and how such properties can be regulated, providing an understanding of the regulated transport of cargos in vivo, starting from single-molecule properties of motors such as dynein in vitro. It begins by describing the functions determined for single-dynein motors in vitro, in a purified and controlled environment. Following this, it turns to the discussion of how the motors function in groups in vitro. Based on this, it reveals the properties emerging from groups of motors and explores which aspects of the single motors are important for their function as a group. Finally, it states that in the last few years, quite a number of factors regulating dynein function have been determined, and combining single-molecule-type approaches to quantifying function in vitro and in vivo with a variety of advanced fluorescence techniques in vivo is likely to allow rapidly accelerating progress in understanding the mechanisms of dynein regulation at a fundamental level.