High-dose methotrexate is a standard component of therapy for high-grade osteosarcoma. Its effectiveness may be limited by intrinsic and acquired resistance. Decreased reduced folate carrier (RFC) expression has been shown in approximately half of osteosarcomas at diagnosis. Mutations and polymorphisms in the RFC gene have been reported in various cell lines. The purpose of this study was to investigate sequence alterations in the RFC gene in osteosarcoma tumor samples. The entire coding region of the RFC gene in samples from 162 osteosarcoma patients was screened by DNA single-stranded conformational polymorphism, followed by direct sequencing of any region with altered mobility. A previously identified polymorphism at cDNA position number 174 of RFC exon 2 was observed. Sixty-one samples (37.6%) were heterozygous with both A/G at this position (His(27)/Arg(27)), 52 samples (32.2%) were homozygous with G (Arg(27)), and 49 samples (30.2%) were homozygous with A (His(27)). Fifteen (9.2%) samples were identified with other RFC sequence variants in exon 2, none of which have been reported. The sequence variants in exon 2 included a G to A substitution at cDNA position 231, a G to A substitution at cDNA position 155, a C to T substitution at cDNA position 114, and a T to C substitution at cDNA position 104, resulting in a serine to asparagine substitution at amino acid 46, a glutamate to lysine substitution at amino acid 21, an alanine to valine substitution at amino acid 7, and a serine to proline substitution at amino acid 4, respectively. A deletion of A at cDNA position 126 resulting in a frameshift was also observed. Some of these variants were observed in multiple samples. Eight samples had altered single-stranded conformational polymorphism patterns in exon 3 that were associated with nucleotide changes that altered the amino acid sequence. All of these RFC sequence variants appeared to be heterozygous. Heterozygous C/T and homozygous C also were observed at RFC cDNA position 790 in exon 3, which does not alter the amino acid coding sequence. This study shows that RFC sequence alterations are frequent in samples from osteosarcoma patients. Additional studies are under way to determine the clinical significance of these sequence alterations and their effect on methotrexate transport and resistance.