The improved outlook for children diagnosed today with acute lymphoblastic leukemia (ALL) over that 40 years ago is remarkable. With modern therapies and supportive care, complete remissions are achieved in up to 95% of patients and long-term disease-free survival rates approach 80%. Methotrexate is a key component in ALL consolidation and maintenance therapies and is administered intrathecally in the prophylaxis and treatment of central nervous system leukemia. Recent reports have significantly extended the results of preclinical studies of methotrexate response and resistance to patients with ALL. The application of new and sensitive molecular biology techniques makes it possible to study specific chromosomal and genetic alterations [t(12;21), hyperdiploidy, deletions or methylation of p15INK4B and p16INK4A] which potentially contribute to methotrexate response and resistance in childhood ALL. Studies of the relationships between genetic alterations and ALL progression, methotrexate pharmacology, and long term event-free-survivals may lead to the better identification of subgroups of patients who exhibit unique levels of sensitivity or resistance to chemotherapy including methotrexate. Further, by characterizing the roles of translocation-generated fusion genes (TEL-AML 1) and tumor suppressor genes (p15INK4B and p16INK4A) in treatment response, it may be possible to identify new and selective targets and/or treatment strategies for both children and adults with ALL who are refractory to current therapies.