Age fractionation of erythrocytes is useful for further studies of the pharmacokinetics of methotrexate (MTX) in red blood cells. We separated erythrocytes from five blood donors and four patients at different time points after MTX infusions, using discontinuous Percoll gradients consisting of four solutions with a difference of 3% in density among them. The procedure yielded five distinct fractions of erythrocytes of increasing mean cell age as judged by declining reticulocyte enrichment and erythrocyte aspartate aminotransferase activity among the five fractions. MTX concentrations of the erythrocytes were measured at different times in connection with five 24-h MTX infusions (0.7-4 g/m2) on 14 occasions. Two days after completion of MTX infusion, no MTX was detected in the youngest erythrocyte population in two patients. Seven days after the infusion, the highest MTX concentrations were found in the youngest red blood cells. Ten to fourteen days following the MTX treatment, considerably lower MTX concentrations were found in the young red blood cells, and the MTX-containing erythrocytes seemed to have moved down the gradient. Just before the next MTX infusion (after 28 days) no MTX could be detected in the young erythrocytes. The MTX concentrations at that time were highest in the oldest erythrocyte fractions. This study shows more directly that MTX is incorporated in the red cell precursors of the bone marrow. The pharmacokinetics demonstrated correspond to a maturation time of the erythroblasts of about 7 days.