The uptake of several folate compounds has been investigated in wild-type and one class of methotrexate-resistant Chinese hamster ovary cells. The wild-type cells can take up methotrexate, folic acid, and 5-methyl tetrahydrofolate. The uptake of methotrexate is characterized by a KT of 1.9 +/- 0.2 microM and a Vmax of 1 +/- 0.8 pmol/min/mg protein, is competitively inhibited by folic acid and 5-methyl tetrahydrofolate, and is sensitive to p-hydroxymercuriphenyl sulfonic acid. The resistant cells are unable to take up methotrexate, folic acid, and 5-methyl tetrahydrofolate. In addition, the resistant cells are unable to bind methotrexate specifically, whereas the wild-type cells bind the drug with an apparent KD of 2 +/- 0.4 microM and a Vmax of 1.3 +/- 0.3 pmol/mg protein. These data indicate that the resistant cells are resistant because of an inability to take up the drug resulting from a defective membrane-binding component. The data also suggest that both methotrexate and folic acid are transported by the same system in Chinese hamster ovary cells.