High‐affinity progesterone‐binding sites have been identified, characterized in and purified from porcine liver membranes. They were functionally solubilized by the non‐denaturing zwitterionic detergent 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonic acid (Chaps, 20 mM, detergent/protein mass ratio 4:1) at a yield of 75–80%. Using [³H]progesterone as radioligand, binding studies showed high‐affinity and low‐affinity binding sites in microsomal preparations with an apparent K_d1 of 11 nM and an apparent K_d2 of 286 nM. In solubilized fractions the high‐affinity binding sites were present at an apparent K_d of 69 nM. In both preparations, progesterone binding was time‐dependent, saturable, reversible, and showed a similar hierachy of affinities for related steroids. A purification scheme was developed based on anion‐exchanger procedures. The purified fraction as identified by maximum specific progesterone‐binding activity contained two major polypeptides of apparent molecular masses (SDS/PAGE) of 28 kDa and 56 kDa, respectively. Sequencing of both polypeptides showed an identical amino terminus without significant identity in the amino acid sequence to any known protein primary structure.