The binding of [3H]1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl propyl) piperazine (GBR-12935), an antagonist of the dopamine transporter, to human P450s expressed in yeast cells was investigated. Among the ten forms of human P450 tested (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, and 3A4), [3H]GBR-12935 bound most strongly to CYP2D6. The calculated Kd of [3H]GBR-12935 binding to CYP2D6 was 42.2 nM, indicating that GBR-12935 has a high affinity for CYP2D6. The characteristics of [3H]GBR-12935 binding to CYP2D6 were investigated by competitive studies using several chemicals. The binding of [3H]GBR-12935 to CYP2D6 was not changed by dopamine, suggesting that these binding sites are not dopamine-sensitive binding sites. The binding of [3H]GBR-12935 to CYP2D6 was decreased partially by substrates or inhibitors of CYP2D isoforms (quinine, quinidine, propranolol, bufuralol, imipramine, and desipramine). By means of binding studies using several forms of expressed human P450, we demonstrated that the CYP2D isoform is one GBR-12935 binding site that is insensitive to dopamine.