Membrane progestin receptors (mPRs) are key mediators of rapid, nongenomic actions of progestins on plasma membranes. We established a procedure for the expression and purification of recombinant goldfish mPRα using the methylotropic yeast Pichia pastoris. In P. pastoris, the recombinant protein, which carried C-terminal histidine and c-Myc tags, was expressed in an active form as the receptor for maturation-inducing steroids of fish. Expressed proteins were bound reversibly with a high affinity (K d = 9.4 nM) at a single binding site that could be saturated. After solubilization of mPRα with n-dodecyl-β-D-maltoside (DDM) from yeast membranes, the recombinant protein was purified using three different columns: first it was affinity-purified over nickelnitrilotriacetic acid (Ni-NTA), then bound to a cellulose resin with free amino groups and finally to a column with affinity for the c-Myc epitope. The identity of the purified protein was verified by MALDI-TOF/MS analysis and its capacity to bind progestin remained. Expression and purification of mPRα protein in its functional form will enable the screening of ligands and the determination of its three dimensional structure.Progestins are important steroids regulating final maturation at reproductive tissues in male and female vertebrates. They can be divided into natural and synthetic types. Natural type is found in humans and certain animals (32). Natural progestin in human is progesterone. In the medical fields, a variety of synthetic progestins are used. In teleost fishes, two distinct progestins have been identified as naturally occurring maturation inducing steroids (MIS), which control oocyte maturation. 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-DHP) was identified in amago salmon (Oncorhynchus rhodurus) and 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S) was identified in Atlantic croaker (Micropogonias undulatus) and spotted seatrout (Cynoscion nebulous) (22). The structural differences between human progestin, progesterone, and fish progestins are that fish progestins have multiple hydroxyl groups on the side chain at positions 17, 20 and 21. In fish, MIS is produced by the follicular envelope in response to luteinizing hormone (LH) from the pituitary (15). In salmon, LH stimulates a number of signaling cascades leading to the production of 17α-hydroxyprogesterone (17α-HP) from progesterone and 17α-hydroxypregnenolone in the theca cells. Then, 17α-HP is converted to 17,20β-DHP by 20β-hydroxysteroid dehydrogenase (20β-HSD) in the granulosa cells (18,22). The membrane progestin receptor (mPR) mediates the nongenomic actions of progestins on the plasma membrane. It was identified in teleost, a ray-finned fish, at first and subsequently identified in other vertebrates including human (44,45). Research into the