Several systems have been developed that allow transcription of a target gene to be chemically controlled, usually by an allosteric modulator of transcription factor activity. An alternative is to use chemical inducers of dimerization, or "dimerizers," to reconstitute active transcription factors from inactive fusion proteins. The most widely used system employs the natural product rapamycin, or a biologically inert analog, as the dimerizing drug. A key feature of this system is the tightness of regulation, with basal expression usually undetectable and induced expression levels comparable to constitutive promoters. In our experiments, the use of the minimal interleukin-2 (IL-2) promoter is an important determinant of this; substitution of a minimal simian virus 40 (SV40) or cytomegalovirus (CMV) promoter results in significantly higher levels of basal expression. The key factor dictating the successful use of the system is achieving high expression levels of the activation domain fusion protein. In the context of clinical gene therapies, the system has the advantage of being built exclusively from human proteins, potentially minimizing immunogenicity in the clinical setting. The dimerizer system has been successfully incorporated into diverse vector backgrounds and has been used to achieve long-term regulated gene expression in vitro and in vivo. This protocol describes the preparation of vectors for rapamycin- or rapalog-inducible gene expression, followed by induction of gene expression in vitro.