Carrier-mediated transport across cell membranes is an important determinant of activity, resistance, and toxicity of chemotherapeutic agents including antiretroviral (ARV) drugs (ARDs). The organic cation transporters (OCTs) 1 and 2 have been implicated in the translocation of different cationic drugs but so far were insufficiently tested for interactions with ARDs. Here, we assessed among cationic drugs commonly used in human immunodeficiency virus (HIV) therapy inhibitors and substrates of OCTs, and analyzed the tissue distribution of OCTs and their expression in lymph nodes (LNs), the primary intracellular target of HIV and ARDs. Inhibitors were identified by measuring the attenuated uptake of the radiolabeled model substrate 1-methyl-4-phenylpyridinium into OCT-transfected human embryonic kidney-293 cells in the presence of ARDs. Substrates were identified by measuring OCT-specific intracellular accumulation using liquid chromatography/tandem mass spectrometry. Inhibitory drugs were (in order of increasing potency): nelfinavir < ritonavir < saquinavir < indinavir < trimethoprim < pentamidine, with consistently lower IC(50) values determined for OCT1. Substrates with highest transport efficacy (V(max)/K(m)) were lamivudine (OCT1, 8 microl/mg protein/min; OCT2, 4.4 microl/mg protein/min) and zalcitabine (OCT1, 4.1 microl/mg protein/min; OCT2, 2.6 microl/mg protein/min). Using quantitative real-time polymerase chain reaction, a marked expression level of OCT1 was detected in human samples of liver, ovary, prostate, and testis, and of OCT2 in kidney, colon, heart, skeletal muscle, and testis. Expression of OCTs in LNs was low in HIV-negative control individuals but dramatically increased in HIV-infected persons. These data suggest that drug interactions about the OCTs may be relevant for the ARV therapy, in particular by influencing drug accession to infected tissues and hepatic or renal elimination.