To treat human immunodeficiency virus (HIV) within the central nervous system (CNS), levels of anti-HIV drugs in the brain must reach therapeutic concentrations. The ability of (-)-2'-deoxy-3'-thiacytidine (3TC; lamivudine) to cross the blood-brain and blood-cerebrospinal fluid (CSF) barriers, alone and in combination with additional nucleoside analogs, was investigated. The bilateral in situ guinea pig brain perfusion method, linked to high-performance liquid chromatography analyses, was used to examine 3TC uptake into brain and CSF simultaneously. The influence of transport inhibitors and additional nucleoside analogs on this uptake was investigated. 3TC movement across the blood-CSF barrier was examined in more detail by the isolated choroid plexus model. 3TC movement across the brain barriers and subsequent accumulation in the brain and CSF was low. However, 3TC uptake from blood into choroid plexus (a potential CNS target for HIV treatment) was significant, and was facilitated by a digoxin-sensitive transporter. Another transporter was identified, which removed 3TC from the choroid plexus. Abacavir, 2'3'-didehydro-3'-deoxythymidine, and 3'- azido 3'-deoxythymidine did not interact with 3TC at either of the brain barriers to affect CNS concentrations of 3TC. However, a significant interaction between 3TC and 2'3'- dideoxyinosine was observed at the choroid plexus, and it may prove beneficial to select drug combinations where no such interaction is indicated.