Apart from fusion inhibitors, 'conventional' antiretrovirals such as nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) act on intracellular targets. Intracellular concentrations of these agents may be an important determinant of antiviral activity, and the pharmacokinetics of intracellular drug accumulation (including binding to cytosolic proteins, intracellular-free fraction, influx and efflux kinetics and intracellular drug metabolism) are likely to impact upon efficacy and toxicity. To date, intracellular drug accumulation has been poorly studied in vivo, due to methodological difficulties and the relatively large volumes of blood required. NRTIs require intracellular conversion to their active phosphorylated metabolites: interactions between these agents, or with other drugs may impact upon efficacy. PIs are metabolised by cytochrome P450 enzymes in gut and liver; some intracellular metabolism by P450 isoforms is also possible. PIs are also substrates for drug efflux transporters such as P-gp and MRP1. We have previously observed a hierarchy of intracellular accumulation of PIs, most probably related to physiochemical characteristics of these drugs such as lipophilicity and plasma protein binding. Comparatively, little is known about the intracellular pharmacokinetics of NNRTIs. These drugs probably do not accumulate inside cells to any significant degree. The study of intracellular pharmacokinetics of HIV drugs is central to investigating putative sanctuary sites where HIV may replicate with little selective pressure. However, stringent methodological procedures need to be applied, and techniques for measuring intracellular drug are in their infancy. Moreover, failure to differentiate between truly intracellular drug and drug bound to cell membranes render results difficult to interpret.