To avoid systemic toxicity of the cytotoxic drug methotrexate (MTX) and to improve tumor selectivity, MTX was bound to human serum albumin (HSA) as a drug carrier.To understand more about the mechanism of action of MTX conjugated to HSA (MTX-HSA), the uptake of MTX-HSA into the cell was determined as well as the effect of MTX-HSA on thymidylate synthase (TS), cell cycle distribution, and cell proliferation.Different uptake kinetics were observed for [3H]MTX and [3H]MTX-HSA.However, similar uptake kinetics were measured for 125I-HSA and 125I-MTX-HSA (2.1 and 1.8 pmol/107 cells/h when cells were treated with 10 μM 125I-HSA and 125I-MTX-HSA, resp.), suggesting that MTX-HSA enters the cells by albumin-mediated endocytosis.We observed no effect of MTX-HSA on TS when folate receptor-expressing KB cells were treated for 4 h (IC50, >50 μM).However, 24 h after incubation, MTX-HSA inhibited TS with an IC50 of 6.9 μM.In addition, we found that MTX-HSA had a delayed effect on the cell cycle compared with MTX and that this effect could be inhibited with the lysosomal inhibitor methylamine, suggesting that MTX-HSA activity is dependent on lysosomal processes.The proliferation of different wild-type and MTX-resistant tumor cell lines was inhibited at IC50 concentrations between 2 and 78 μM, resp.MTX-HSA accumulates in vivo in the tumor tissue.Local concentrations of 18-29 μM were measured, which are effective anti-proliferative concentrations as determined in vitro.We also investigated the antitumor activity of MTX-HSA in vivo in different human tumor xenografts grown s.c. in nude mice.Fourteen tumors from eight different tissues were tested.Nine of 14 tumors (64%) showed a clear response with tumor inhibition, stasis, or regression; 5 of 14 (36%) gave a moderate response with tumor growth delay or no response.In conclusion, MTX-HSA is effectively taken up by the cells via albumin receptor- or folate receptor-mediated endocytosis and time-dependently released as an active compound into the cytosol to exert an inhibiting effect on TS and to induce cell cycle alterations.In vivo, effective concentrations of MTX-HSA were reached in tumor tissue to exhibit antitumor activity.