We recently found that phosphatidyl-2',3'-dideoxycytidine (phosphatidyl-ddC) had substantial anti-hepatitis B virus (HBV) activity in vitro compared to 2',3'-dideoxycytidine (ddC) (Hostetler et al. (1994) Antiviral Res. 24, 59-67). Upon administration of liposomal phosphatidyl-ddC to mice, a 40-fold higher drug area under curve was observed in the liver. To evaluate the possibility of using liver-targeted anti-HBV nucleosides to treat woodchuck hepatitis virus, we wanted to find the most potent and selective lipid conjugates. It has been shown that 2',3'-dideoxy-3'-thiacytidine as a racemic mixture of the cis-isomer (cis-(+/-)-BCH-189) has much greater activity against HBV viruses than ddC in vitro. Recently, it was shown that the (-)-beta-L-enantiomer (3TC) is more active and less toxic than the (+)-beta-D-form ((+)-BCH-189). To determine whether phospholipid conjugates of 3TC retain antiviral activity in 2.2.15 cells as demonstrated previously with ddC, we synthesized the 1,2-dipalmitoyl-sn-glycerol-3-phosphate conjugates of (+/-)-BCH-189 and 3TC and assessed their anti-HBV and anti-HIV activities, in vitro. Phosphatidyl-3TC and phosphatidyl-BCH-189 had antiviral activity comparable to the respective free drugs in 2.2.15 cells which chronically produce HBV. In HIV-1-infected human peripheral blood mononuclear cells and HT4-6C cells, phosphatidyl-3TC and phosphatidyl-(+/-)-BCH-189 exhibited significantly lower activity than the corresponding free nucleosides. In view of the documented ability of phosphatidyl-ddC to target drug to the liver, it seems reasonable to expect that phosphatidyl-3TC or phosphatidyl-(+/-)-BCH-189 could be employed to provide greatly enhanced hepatic antiviral activity in HBV infection in vivo.