Active transport across biological membranes represents a critical step in the disposition of many drugs. It is now well-established that different efflux and uptake transporters such as P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs) or organic anion transporting polypeptides (OATPs) are involved in the overall disposition and efficacy of numerous compounds. These proteins are mainly expressed at physiological sites of drug absorption and elimination, thus leading to diminished absorption and/or increased transporter-facilitated excretion. Moreover, drug transporters are known to be of protective significance in blood-organ barriers. On the contrary, only little is known about the relevance of transporter function on drug levels within tissues and cellular compartments, i.e. the site of action for many substances. Moreover, the pharmacokinetic processing inside the cell is characterized by uptake, metabolism and elimination. It is gradually being recognized that active uptake and/or efflux transporters may modify target concentrations at the subcellular receptor sites which in turn may have an influence on drug effects. This review will summarize current knowledge about the impact of transporter proteins on drug availability within pharmacologically relevant cellular compartments and tissues as hepatocytes, enterocytes, different blood cell types, brain, and the heart with emphasis on the potential clinical significance of these transporters.