Many clinical studies have been conducted on the role of high-dose methotrexate (MTX) in human osteosarcoma, but information about the in vitro effect of MTX on human osteosarcoma cells is lacking. The effect of MTX on tumor cells derived from 7 patients with osteosarcoma was studied in an attempt to correlate clinical and in vitro sensitivity to the drug. Isolation of the cells from the primary tumors (4 patients) or metastasis (3 patients) was carried out with a collagenase treatment followed by purification through a density gradient. The osteosarcoma cells were identified by EM and histochemical reactions. The cellular sensitivity to MTX was measured by the inhibitory effect of MTX on [3H]deoxyuridine incorporation into DNA. This incorporation was 50% inhibited in primary tumors at concentrations from 3 .times. 10-7 to 3 .times. 10-6 M. The metastatic cells isolated from patients that were clinically resistant to high-dose MTX had a 50% inhibition ranging from 1.5 .times. 10-7 to 4 .times. 10-5 M. Human stimulated lymphocytes, [mouse] sarcoma 180 cells and Ehrlich ascitic mouse [tumor] cells had a 50% inhibition of .apprx. 1.5 .times. 10-7 M. When [3H]thymidine incorporation into DNA of human osteosarcoma cells was studied, MTX increased its incorporation up to 4-fold. This increase was stable for at least 6 h and was only slightly enhanced by the addition of hypoxanthine. The stimulation by MTX of [3H]thymidine incorporation into DNA in stimulated lymphocytes and Ehrlich cells is much smaller, between 40-60%. Osteosarcoma cells probably build their deoxythymidine monophosphate pool largely through the de novo pathway, the salvage pathway being less important. The importance of the de novo pathway for deoxythymidine monophosphate synthesis probably is a biochemical characteristic of the osteosarcoma cells which could be related to the initial sensitivity of this tumor to MTX; an activation of the salvage pathway probably could constitute an additional mechanism of resistance to this drug.