Summary: Antifolates were the first metabolic antagonists that were brought to the clinical practice about sixty years ago. By that time it was known that aminopterin and its methylderivative, methotrexate, can interfere with folate metabolism by inhibiting the enzyme dihydrofolate reductase. The discovery of polyglutamylation that came during the eighties brought a breakthrough in understanding both physiology of natural folates and pharmacology of folate inhibitors as well. Another important milestone was elucidation of interactions of antifolates with its target enzyme, dihydrofolate reductase, and discovery of folate transport processes. By that time methotrexate was already a standard component of a number of chemotherapy regimens used in the clinic. In pediatric oncology, where the high-dose administration schemes have been sucessfully used, toxicity and mechanisms of resistance emerged as significant problems that often limit clinical use of folate antagonists.