Methotrexate (MTX) and 6-mercaptopurine (6-MP) are used for maintenance therapy of acute lymphoblastic leukemia (ALL) of childhood, and both are myelotoxic. Clinically the individual tolerance to the two drugs is variable. In order to study to what extent the MTX accumulation in circulating neutrophils is related to the absolute neutrophil count (ANC), neutrophils were isolated on a discontinuous two-step Percoll gradient in 16 children with ALL in maintenance therapy. The MTX concentration in the neutrophils was determined with a sensitive radioligand binding assay. In all children except one who admitted noncompliance, MTX was found in the neutrophils in concentrations (56-460 pmol/10(9) cells) positively correlated with the weekly dose of MTX (r = 0.51, p less than 0.01). The interindividual variation was large. Children with relatively low neutrophil MTX exhibited the widest intraindividual variation of neutrophil MTX upon reexamination during continued MTX administration with the same dosage schedule. Increases in the weekly dose of MTX resulted in proportional increases in the neutrophil MTX. In half the cases the ANC was less than 1.5 X 10(9)/l. The ANC was not related to the weekly MTX dose or the daily 6-MP dose. In children with ANC greater than 1.5 X 10(9)/l there was a significant inverse correlation between the ANC and the neutrophil MTX (r = -0.71, P less than 0.01), which was not found in the group of children with ANC less than 1.5 X 10(9)/l. These findings may be explained by differences in the kinetics of the granulopoiesis between children with high and children with low neutrophil counts.