Current standard-of-care regimens recommended for the treatment of HIV infection include two or more nucleos(t)ide reverse transcriptase inhibitors (NRTI) in combination with a protease or non-nucleoside reverse transcriptase inhibitor. NRTIs are activated through interactions with the cellular machinery for regulating endogenous nucleoside triphosphate (NTP) pools. Once activated to their triphosphate form, NRTIs compete with natural 2'-deoxynucleoside triphosphates (dNTP) for incorporation by the virally encoded reverse transcriptase and host polymerases. Competitive inhibition, changes in enzyme expression, or allosteric modulation of cellular metabolizing enzymes may therefore alter NRTI activation or perturb cellular dNTP levels causing changes in NRTI antiviral activity and toxicity. This paper reviews the unique metabolic profiles of NRTIs and discusses methodologies for understanding the effects of combining them. Cell culture experiments assessing the antiviral synergy and intracellular metabolism of NRTI combinations have yielded valuable insights into the behavior of treatment regimens in vivo. The development of more reliable and convenient methods for detecting nucleotides, including those applying mass spectrometry, are helping to further elucidate the intracellular pharmacology of NRTIs. Studies assessing the potential for intracellular NRTI drug-drug interactions will facilitate a better understanding of the efficacy of current therapies, as well as the design of combination therapies with optimal activity and toxicity profiles.