An L1210 cell line showing a 300-fold resistance to the cytostatic effect of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) (designated L1210/PMEA-1) was selected in cell culture upon exposure of wild-type L1210/0 cells to stepwise-increased drug concentrations. The mutant L1210/PMEA-1 cell line was characterized by an unusual specificity in that the cytostatic activity was severely impaired only for PMEA and the closely related 2,6-diaminopurine derivative PMEDAP, but not for its guanine counterpart PMEG or for 9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA). The L1210/PMEA-1 cell line showed poor resistance to the cytostatic activity of the lipophilic PMEA prodrug bis(POM)PMEA and virtually kept its PMEA resistance profile in the presence of indomethacin, excluding resistance of the cells of PMEA and PMEDAP by an increased efflux of the drugs. Intracellular purine nucleotide pool labelling studies with adenine, hypoxanthine and glycine revealed that PMEA/PMEDAP resistance did not originate from a defect in the enzymatic pathways of purine nucleotides. ATP, AMP and cAMP, but not adenosine, adenine, HPMPA and inhibitors of nucleoside transport carriers markedly interfered with PMEA uptake in L1210/0 cells. The L1210/PMEA-1 cells proved to have less than 10% of the PMEA uptake capacity of wild-type L1210/0 cells as measured by rapid sampling kinetics as well as long-term incubation experiments. After a 24-h incubation period, the intracellular levels of [2,8-3H]PMEA and its phosphorylated metabolites were approximately 10-fold lower in L1210/PMEA-1 cells than in L1210/0 cells. Our observations point to a compromised and highly specific PMEA/PMEDAP uptake as the molecular basis for the pronounced PMEA resistance of the mutant L1210/PMEA-1 cells.