The emergence of resistant hepatitis B virus (HBV) with the L528M mutation and/or the M552V and M552I mutations in the polymerase gene following long-term lamivudine treatment is becoming an important clinical problem. The aim of this study was to investigate the susceptibility of wild-type and lamivudine-resistant HBV to MCC-478 (LY582563), a novel nucleoside analogue derivative of phosphonomethoxyethyl purine. The susceptibility of wild-type HBV and lamivudine-resistant mutants (M552I, M552V, and L528M/M552V) to MCC-478 was examined by transient transfection of full-length HBV DNA into human hepatoma cells. HBV DNA replication was monitored by Southern blot hybridization, and the effective concentration required to reduce replication by 50% (EC(50)) was determined. The replicative intermediates of wild-type and lamivudine-resistant mutants were progressively diminished by treatment with increasing doses of MCC-478. The MCC-478 EC(50)s were 0.027 microM for wild-type HBV (about 20 times more efficient than lamivudine), 2.6 microM for M552I, 3.3 microM for M552V, and 2.0 microM for L528M/M552V. Wild-type HBV and lamivudine-resistant mutants are susceptible to MCC-478. MCC-478 appears to be a candidate for the treatment of HBV infection and exhibits potent activity against lamivudine-resistant HBV.