This study aimed to purify, clone, and express the mammalian sigma1-binding site, a protein that binds to sigma-ligands such as haloperidol, pentazocine, and ditolyguanidine. The sigma1-binding site was purified from guinea pig liver microsomes using (+)[3H]pentazocine and (−)[3H]azidopamip as specific probes. The purified protein retained high affinity for these ligands and showed no homology to known proteins after partial amino acid sequencing. Radiation inactivation of the pentazocine-labeled site yielded a molecular mass of 24 ± 2 kDa. The corresponding cDNA was cloned using degenerate oligonucleotides and cDNA library screening. The open reading frame encoded a 25.3-kDa protein with at least one putative transmembrane segment. The protein expressed in yeast cells transformed with the cDNA showed pharmacological characteristics similar to the brain and liver sigma1-binding site. The deduced amino acid sequence was structurally unrelated to known mammalian proteins but shared homology with fungal proteins involved in sterol synthesis. Northern blot analysis revealed high densities of the sigma1-binding site mRNA in sterol-producing tissues, supporting the known ability of sigma1-binding sites to interact with steroids like progesterone. The study provides insights into the functional role of the sigma1-binding site and its potential involvement in sterol metabolism.This study aimed to purify, clone, and express the mammalian sigma1-binding site, a protein that binds to sigma-ligands such as haloperidol, pentazocine, and ditolyguanidine. The sigma1-binding site was purified from guinea pig liver microsomes using (+)[3H]pentazocine and (−)[3H]azidopamip as specific probes. The purified protein retained high affinity for these ligands and showed no homology to known proteins after partial amino acid sequencing. Radiation inactivation of the pentazocine-labeled site yielded a molecular mass of 24 ± 2 kDa. The corresponding cDNA was cloned using degenerate oligonucleotides and cDNA library screening. The open reading frame encoded a 25.3-kDa protein with at least one putative transmembrane segment. The protein expressed in yeast cells transformed with the cDNA showed pharmacological characteristics similar to the brain and liver sigma1-binding site. The deduced amino acid sequence was structurally unrelated to known mammalian proteins but shared homology with fungal proteins involved in sterol synthesis. Northern blot analysis revealed high densities of the sigma1-binding site mRNA in sterol-producing tissues, supporting the known ability of sigma1-binding sites to interact with steroids like progesterone. The study provides insights into the functional role of the sigma1-binding site and its potential involvement in sterol metabolism.