When embarking upon any transfection procedure, a critical first step is to optimize conditions. Every mammalian cell type has a characteristic set of requirements for optimal introduction of foreign DNA; there is a tremendous degree of variability in the transfection conditions that work, even among cell types that are very similar to one another. Often, an experimenter must screen a wide variety of cell types for a desired regulatory trait, such as an appropriate response to a particular effector molecule. It is thus helpful to have a straightforward, systematic approach to optimizing transfection efficiency. This unit provides guidelines to optimizing transfection experiments.