• Are multiple intracellular targets better than one? Does inhibition of dihydrofolate reductase (DHFR), thymidylate synthase (TS) or enzymes of the purine biosynthetic pathway confer an advantage? Alternatively, is inhibition of purine biosynthesis a safety liability rather than an advantage? • Can a lipophilic molecule rather than one that uses the folate carrier mechanisms, i.e. the folate binding protein (FBP) and or the reduced folate carrier (RFC), circumvent intrinsic or acquired resistance? • Can tumor selectivity be increased by exploiting knowledge of different patterns of RFC versus FBP expression or of variations in FBP isoform expression? • How does a patient’s functional folate status or supplemental folic acid modulate efficacy and or toxicity? • Does an antifolate that is a good substrate for the intracellular enzyme folylpolyglutamate synthase (FPGS) have a selective advantage? Does polyglutamation of the antifolate confer a therapeutic advantage because it results in increased potency against the target enzyme, and increased polarity leading to increased cellular retention? Alternatively, is it a disadvantage with respect to safety of the molecule because of prolonged intracellular retention times or the potential for resistance? • Does an increase in the potency of binding to the target enzyme confer a therapeutic advantage? • Resistance mechanisms include decreased cellular accumulation due to impaired transport, decreased retention inside the cell as a consequence of a lack of polyglutamate formation, or an increase in gamma glutamyl hydrolase (GGH) activity, an increase in expression of the target enzyme, or a mutation in the target enzyme. Which of these is clinically relevant, and how does knowledge of these resistance mechanisms aid selection of the appropriate antifolate for treatment of an individual patient? • How do pharmacokinetic variables contribute to safety and efficacy? Do the newer antifolates have fewer propensities for drug-drug interactions? • Is there an intrinsic limit to the clinical activity of an antifolate as a single agent? Can antitumor activity be increased, or can an increase in activity only be achieved by combining antifolates with other anticancer drugs (either other antifolates which target different folate requiring enzymes or compounds with different mechanisms of action)? Similarly, can the spectrum of clinical activity be increased? • Can patients whose tumors have a specific set of molecular properties be identified prospectively, and matched with the pharmacological properties of an agent, i.e. can therapy of individual patients be tailored? • Can toxicity be decreased and quality of life improved? • Can patients at risk of serious toxicity to a given agent be identified before or during therapy and managed in the light of that knowledge? • Are there mechanisms for rescuing patients from toxicity, and not from antitumor activity? • What improvements can be made in the administration schedule and therefore convenience to patients?