Plasma concentrations of 7-hydroxymethotrexate were measured in 10 individuals receiving methotrexate, 8 of them per infusion. After the end of infusion, the plasma concentration of formed 7-hydroxymethotrexate fell less rapidly than that of methotrexate. At 24 h after infusion, plasma concentration ratios of 7-hydroxymethotrexate ranged from 30 to 1 (n = 8). The presence of 7-hydroxymethotrexate was shown to influence the transport of methotrexate into Ehrlich ascites tumor cells and human KB cells, cultured in vitro. These findings suggest that monitoring of 7-OHMTX is important in MTX treatment.