The recent availability of high specific activity radiolabeled dopaminergic antagonists with specificity for dopamine receptor subtypes has allowed us to screen a wide variety of cultured mammalian cell lines for the presence of D1 and D2 dopamine receptors. Specific receptor binding of the D1 selective antagonists [3H]SCH 23390 and [125I]SCH 23982 was detected in membranes prepared from NS20Y cells, a clonal cell line derived from the C1300 murine neuroblastoma. Saturation analysis of [3H]SCH 23390 binding revealed the presence of saturable, high affinity binding sites with a dissociation constant (Kd) of 575 pM and a receptor density of 138 fmol/mg protein (approximately 9000 receptors/cell). Inhibition of [3H]SCH 23390 binding by a series of dopaminergic agonists and antagonists exhibited appropriate stereoselectivity and pharmacological specificity, verifying the D1 nature of this site. Dopamine inhibition of [3H]SCH 23390 binding revealed the presence of high and low affinity agonist binding sites which were converted to a homogeneous low affinity state by the addition of GppNHp. In membranes prepared from the WERI 27 human retinoblastoma cell line, specific receptor binding of the D2 antagonists [3H]methylspiperone and [125I]NAPS was observed. Saturation analysis of [3H]methylspiperone binding revealed the presence of a single class of high affinity, saturable binding sites with a Kd of 140 pM and a Bmax of 223 fmol/mg protein (approximately 2500 receptor sites/cell). Inhibition of [3H]methylspiperone binding by dopaminergic antagonists exhibited a rank order of potency consistent with the identification of a D2 dopamine receptor subtype. In addition, dopamine inhibition of [3H]methylspiperone binding exhibited both high and low affinity agonist binding sites which were converted to low affinity by the addition of GppNHp. These results represent the first direct demonstration of D1 and D2 dopamine receptors in cultured mammalian clonal cell lines. These cells should provide powerful model systems for investigating the molecular mechanisms involved in dopamine receptor/effector coupling and regulation.