We have studied by flow cytometry the transport of fluorescein-methotrexate in Chinese hamster ovary cells. Fluorescein-methotrexate appears to enter cells via a mechanism different from the carrier-mediated system for methotrexate. This conclusion is supported by the following observations: 1) Fluorescein-methotrexate is transported equally well into normal and mutant cells defective in the inward methotrexate uptake. 2) Folic acid and its reduced states, which competitively inhibit methotrexate uptake, do not alter fluorescein-methotrexate transport. 3) Fluorescein-methotrexate accumulation exhibits a low temperature coefficient (Q10 = 1.6) compared with the influx of methotrexate (Q10 = 6-8). 4) Initial rates of fluorescein-methotrexate uptake are concentration dependent but are not saturable. 5) Fluorescein-methotrexate uptake is very slow and reaches steady state after 8 h, whereas at an equimolar concentration methotrexate reaches saturation after 20 min. 6) Initial influx rates of fluorescein-methotrexate are not affected by the presence of methotrexate. 7) Sulfhydryl-reactive mercurials, which block methotrexate transport, do not reduce fluorescein-methotrexate influx, but rather stimulate it. Thus, based on the nonsaturability of fluorescein-methotrexate inward transport, its low temperature coefficient, and lack of inhibition with structural analogs, we conclude that fluorescein-methotrexate is accumulated in hamster cells by a passive diffusion process.