P-glycoprotein (P-gp), encoded by the (ATP-binding cassette transporter superfamily B member 1) gene, is a transport protein involved in the efflux and distribution of the osteosarcoma drugs methotrexate, doxorubicin, actinomycin D, and etoposide. studies indicate a close relationship between the (C1236T) single-nucleotide polymorphism (SNP) and the efficacy of these drugs. The purpose of this research was to elucidate the effect of (C1236T) polymorphism on P-gp-mediated efflux of osteosarcoma drugs. Two stable recombinant Caco-2 cell lines were generated by transfection with either the wild-type allele or the variant allele. The two cell lines were compared in terms of drug resistance, intracellular accumulation, and efflux of methotrexate, doxorubicin, actinomycin D, and etoposide. Accumulation of methotrexate, doxorubicin, actinomycin D, and etoposide was significantly lower in cells overexpressing wild-type P-gp than in untransfected control cells, indicating that these drugs are substrates of P-gp. Actinomycin D accumulated to similar extents in cells overexpressing wild-type or variant P-gp. Methotrexate and etoposide were transported to a greater extent by variant P-gp than wild-type protein. Conversely, doxorubicin was transported to a greater extent by wild-type P-gp. The (C1236T) polymorphism affects P-gp-mediated transport of osteosarcoma drugs in a drug-specific way. These studies support the importance of the (C1236T) SNP for P-gp activity and its potential to explain the alterations in drug response.