Cytochrome P450 3A4 (CYP3A4), an enzyme that is highly expressed in the human liver and small intestine, plays a major role in the metabolism of a large variety of xenobiotics, including an estimated 50% of therapeutic drugs, as well as many endogenous compounds. The expression of CYP3A4 can be induced by xenobiotics. Such induction leads to accelerated metabolism of the xenobiotics themselves (autoinduction) or of concomitantly administered CYP3A4 substrates/drugs, thereby significantly altering their pharmacokinetic and pharmacodynamic profiles. During the past decade, much progress has been made in our understanding of the biological mechanisms responsible for regulation of CYP3A4 expression. It is now known that many xenobiotics induce CYP3A4 expression via the pregnane X receptor (PXR) pathway, while others are thought to act through the constitutive androstane receptor (CAR) and the vitamin D receptor (VDR). As a result, most pharmaceutical companies have recognized that it is important to evaluate CYP3A4 induction potential preclinically and are using primary cultures of human hepatocytes and/or PXR reporter gene assays. In general, the results from these two assay methods correlate well. The reporter gene assays in particular can be used to rapidly screen hundreds of drug candidates, whereas methods using primary human hepatocyte cultures may more accurately assess the potential for CYP3A4 induction in vivo. Although it is important to consider CYP3A4 induction in the early stages of the drug development process, it should be recognized that the assessment of induction potential preclinically is a difficult and imprecise endeavor and can be complicated by many factors.