The lymph node targeting ability and pharmacokinetics of methotrexate (MTX) after intramuscular (i.m.) injection of differently charged liposome-entrapped [H-3]MTX to rats were evaluated using [H-3]MTX as a tracer. Neutral liposomes were prepared with a mixture of phosphatidylcholine, cholesterol and alpha-tocopherol (8:4:0 . 1, molar ratio). Positively and negatively charged liposomes were also prepared by incorporation of stearylamine (8:4:0 . 1, molar ratio) and dicetylphosphate (8:4:0 . 1:1, molar ratio) into neutral liposomes respectively. The encapsulation efficiency (as expressed in terms of radioactivity) in liposomes was increased as alpha-tocopherol was incorporated into the lipid bilayer. The disappearance of [H-3]MTX from the i.m. injection site was rapid and essentially complete after 30 min. On the other hand, the disappearance of radioactivity of liposome-entrapped [H-3]MTX was much slower when compared to free drug. The area under the drug concentration-time curve (AUC) of liposome-entrapped [H-3]MTX in lymph nodes was significantly increased when compared to free [H-3]MTX. It suggested that liposomes injected by the i.m. route entered into the lymphatics and only drug released from liposomes diffused directly into the systematic circulation. The liposome-entrapped [H-3]MTX by i.m. route was markedly localized in the lymph nodes. The concentration of MTX-equivalents in regional lymph node after i.m. injection of liposome-entrapped [H-3]MTX was > 100-350 fold higher when compared to the plasma concentration. These values are more than 10-20 fold higher compared to the i.m. injection of free [H-3]MTX. The positively charged liposomes were more localized in lymph nodes compared to neutral and negatively charged ones. While liposomes injected by i.v. route were localized in liver, spleen and lung compared to free [H-3]MTX, it was evident that i.m. administration of liposomes resulted in enhanced localization of MTX in the lymphatic system but decreased deposition in kidney, liver and other non-targeting tissues compared to free [H-3]MTX. The targeting ability and carrier properties of liposome-entrapped anticancer drugs with varying surface charge, lipid compositions and route of administration are of significant importance to alter biodistribution in chemotherapy.