Microinjection is an excellent technique for studying the acute responses of cells to proteins and can be used to investigate the effects of mutations in proteins on their activity. It has been used widely to study the responses to Rho family guanosine triphosphatases and is particularly useful for cell types that are difficult to transfect. Here, we describe the procedure for microinjecting cells with purified recombinant proteins or with expression vectors encoding proteins, and for analyzing the cells after injection.