Abstract: Rapid exchange of the medium surrounding cells is an essential tool for many cell physiological experiments. Typical microscope perfusion chamber designs represent a compromise between optical considerations, flow requirements, and shear stress on the cells; optical constraints imposed by DIC microscopy are often favored at the expense of flow characteristics necessary for rapid exchange of media. We have developed a perfusion chamber which enables fast, reproducible medium exchange under controlled shear conditions. Our chamber is best suited for high numerical aperture epifluorescence microscopy and phase contrast microscopy with a long working distance condenser, but it cannot be used for DIC microscopy. Complete exchange of the medium over a 12 mm diameter area of the cover slip can be accomplished in several seconds, and the medium over a typical microscope field can be exchanged in about 1 second, with shear rates that will not damage adherent cells (0.1 - 3.0 dyn/cm2). The combination of this chamber with the Flow Injection technique, which is widely used in analytical chemistry, provides a highly versatile tool for microscopy. Experiments requiring complex perfusion profiles may be run under computer control with better than 1% reproducibility of the concentration profile from run to run. This technique is a significant improvement for cell physiological experiments requiring rapid, precise environmental control on the microscope stage. (Supported by N.I.H. grant SSS-3 (S) ROl GM 45260-2.)