Publisher Summary This chapter reviews the method of direct needle microinjection and discusses the application of this method in the study of mitotic cells. As virtually any molecule of interest that can be purified and obtained at an appropriate concentration can be injected into the living mitotic cell, this method can be utilized to study how diverse molecules participate in the events of mitosis. Microinjection of mitotic cells combines high-resolution light microscopic analysis of mitotic events in the living cell with the ability to selectively modify and/or monitor specific molecules. Mitotic cells can be injected with function blocking antibodies and various inhibitors or toxins to determine the contribution of the targeted molecules to mitosis. A recent report also demonstrates that microinjected mRNA molecules can be translated in mitotic cells, thus providing an additional means to modify the molecular composition of the injected cell. In addition to inhibiting the normal function of target molecules, microinjection also makes it possible to monitor the behavior and distribution of various molecules during mitosis.