We have cloned a σ receptor from rat brain and established its functional identity using a heterologous expression system. The cloned cDNA (1,582 bp long) codes for a protein of 223 amino acids that possesses a single putative transmembrane domain. The amino acid sequence of the rat brain σ receptor is highly homologous to that of the σ receptor recently cloned from guinea pig liver and a human placental cell line but is not related to any other known mammalian receptors. When expressed in HeLa cells, the rat brain σ receptor cDNA leads to a two- to threefold increase in haloperidol binding, and this cDNA-induced binding is sensitive to inhibition by several σ receptor-specific ligands. Kinetic analysis using the heterologous expression system has revealed that the rat brain σ receptor interacts with haloperidol with an apparent dissociation constant (KD) of 3 nM. Functional expression of the cloned rat brain σ receptor in HeLa cells also leads to an increase in the binding of two other σ ligands, namely, (+)-pentazocine and (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (PPP). Pharmacological characterization of the cloned rat brain σ receptor reveals that it exhibits severalfold higher affinity for clorgyline than for 1,3-di(2-tolyl)guanidine, it interacts with progesterone and testosterone, and its interaction with PPP is markedly enhanced by phenytoin. In addition, transfection of MCF-7 cells, which do not express type 1 σ receptor mRNA or activity, with the cloned rat brain cDNA leads to the appearance of haloperidol-sensitive binding of (+)-pentazocine, a selective type 1 σ receptor ligand. These data show that the cloned rat brain cDNA codes for a functional type 1 σ receptor. Northern blot analysis with poly(A)+ RNA isolated from various rat tissues has indicated that the σ receptor-specific transcript, 1.6 kb in size, is expressed abundantly in liver and moderately in intestine, kidney, brain, and lung.