A cDNA clone (S2-1a) isolated from a mouse brain cDNA library, using a guinea pig sigma1 cDNA as probe, has high homology to the predicted protein sequence of the guinea pig (88%) and human (90%) sigma1 receptors. Northern analysis revealed a major mRNA of approximately 1.8 kb in a wide range of mouse tissues, with highest levels in brain, liver, kidney, and thymus. Southern analysis and chromosomal mapping in the mouse suggested a single-copy gene in region A5-B2 of chromosome 4. Expression of the clone in MCF-7 and CHO cells led to a pronounced increase in (+)-[3H]pentazocine binding with a selectivity profile consistent with sigma1 receptors. In vitro translation yielded a protein of approximately 28 kDa, as did transfection of a probe containing the hemagglutinin (HA) epitope (S2-1a.HA) into CHO cells, as determined by western analysis using an antibody directed against HA. (+)-[3H]-Pentazocine binding to immunopurified HA-tagged receptor demonstrated conclusively that S2-1a.HA encodes a high-affinity (+)-[3H]pentazocine binding site with characteristics of a murine sigma1 receptor. An antisense oligodeoxynucleotide designed from S2-1a potentiated opioid analgesia in vivo.