4-Amino-pteroyl-glutamic acid (Aminotrexate; AMT) has several advantages over the related antifolate methotrexate (MTX), including greater potency, complete oral bioavailability, and greater accumulation by leukemic blasts in vitro. We compared the pharmacodynamic properties of AMT (given orally at 4 mg/m2 in two divided doses per week) and MTX (100 mg/m2 in four divided doses per week) among children with acute lymphoblastic leukemia. We find AMT and MTX to have equivalent penetration into the bone marrow compartment of these patients, as indicated by the steady-state concentrations within mature red blood cells (RBCs). However, MTX concentrations in the cerebrospinal fluid after oral dosage are significantly greater than AMT. To confirm these clinical observations, mice were treated four weekly injections of AMT or MTX, at a 1:20 dosage ratio, and tissue antifolate content was then determined over the subsequent 22 days. We confirm the selective exclusion of AMT from the CNS compartment, while showing equivalent accumulation of AMT and MTX in the RBCs, liver, spleen, kidneys and testes. Finally, we demonstrate that AMT, MTX, and their predominant polyglutamate species are equipotent inhibitors of their target intracellular enzyme dihydrofolate reductase, emphasizing the critical nature of steady-state tissue accumulation in determining the relative cytotoxic potency of these two antifolates.