High-dose methotrexate is a major component of cur-rent protocols for the treatment of osteosarcoma, but some tumors seem to be resistant. Potential mechanisms of resist-ance include decreased transport throughthe reduced folate carrier (RFC) and increased expression of dihydrofolate reductase (DHFR). To investigate methotrexate resistance, tumors were obtained from 42 patients with high-grade osteosarcoma. RFC and DHFR mRNA expression were studied by semiquantiiative reverse transcription-PCR. The PFC and DHFR genes were studied for deletions and am-plification bySouthern blot. Thirteen of 20 (65%) osteosar-coma samples were found to have decreased RFC expression at the time of intial biopsy. At definitive surgery and re-lapse, 10 of 22 (45%) were found to have decreased RFC expression. Seventeen of 26 (65%) samples with a poor response to chemotherapy had decreased RFC expression, response to chemotherapy had decreased RFC expression, whereas 5 of 14 (36%) samples with a good response had a decrease (p=0.03). None of the samples had an RFC gene deletion. Two of 20 samples (10%) showed increased DHFR expression at inital biopsy. The frequency of increased DHFR expression was significanfly higher inmetastatic or recureent tumors (62%, p=0.014). None of the samples showed evidence of DHFR gene amplification. The high frequency of decreased RFC expression in the biopsy mate-rail suggests that impaired transport of methotrexate is a common mechanism of intrinsic resistance in osteosarcoma. Increased DHFR expression in the pulmonary metastases may be a mechanism of acquired methotrexate resistance or a difference between primary and metastatic lesions.