We report here a procedure for the production in Escherichia coli and subsequent purification and characterization of an 80-residue fragment of the human A-opioid receptor. The fragment (dTM2-3T), which comprises the second and third transmembrane segments as well as the first extracellular loop of the receptor, was expressed as a fusion with glutathione-S-transferase. The fusion protein, which accumulated in insoluble inclusion bodies, was solubilized with N-lauroylsarcosine, and TM2-3 was obtained by thrombin cleavage of the fusion protein followed by reversed-phase HPLC purification. CD spectroscopy of TM2-3 in lysophosphatidylcholine micelles showed that TM2-3 adopts ~50% a-helical structure in this environment, with the remainder consisting of disordered and/or hstructure. This is consistent with the assumption of an a-helical structure by the two membrane-spanning regions and a nonhelical structure in the loop region of TM2-3. Fluorescence spectroscopy and fluorescence quenching experiments suggested that the extracellular loop lies near the surface of the lysophosphatidylcholine micelle. Our work shows that the study of large receptor fragments is a technically accessible approach to the study of the structural properties of the A-opioid receptor and, possibly, other Gprotein-coupled receptors as well.