We describe here a second generation apparatus for studying transient reaction conformations in macromolecules and their complexes by electron cryo-microscopy. Reactions are trapped by rapid freezing in times ranging from a few milliseconds to tens of seconds after initiation. Blotting of the electron microscope grid and freezing it in liquid ethane uses computer controlled microstepping motors. For the fastest time resolution, a blotted grid containing a thin film of one reactant is sprayed with small droplets containing a second reactant just before freezing. The spray is produced electrically (electrospray), which gives a dense cloud of droplets <1μm in diameter from the 1-2μl of solution required per grid. A second method in which two solutions are first mixed by turbulent flow and then blotted prior to freezing is used for reactions with time courses >1s. © 2003 Elsevier Inc. All rights reserved.