INTRODUCTIONGFP (green fluorescent protein) fusion proteins have been used to address a wide range of questions in individual cells, as well as in tissues of a particular organism. GFP fusion proteins can be transiently or stably expressed. Although transient expression is quick and can provide informative results, in many cases it is beneficial and/or essential to develop stable cell lines expressing the fusion protein of interest. In addition to providing more native levels of expression, individual clones can be generated from single cells, the integration site of the plasmid mapped, and the copy number determined. Because every cell in the population is expressing the fusion protein, cell cycle analyses and biochemical fractionation are significantly easier to accomplish. The following protocol can be used for the development of stable cell lines expressing GFP fusion proteins. Although optimal transfection procedures (e.g., calcium phosphate, electroporation, or FuGENE 6 [Roche Applied Science]) vary depending on cell type, this general transfection procedure has been successful for stable transfection of HeLa, A-431, U2OS, BHK, and HT1080 cells.