This study demonstrates the presence of [3H]-progesterone binding protein on the cell surface of human spermatozoa. The binding protein is masked and is exposed after treatment of spermatozoa with surfactants such as digitonin. Specific binding of [3H]-progesterone is observed after the washed spermatozoa or the crude cell membrane fractions are treated with 0.1% digitonin at 0-4 degrees C for 30 min R5020, 17alpha-hydroxyprogesterone and testosterone only partially compete with [3H]-progesterone for binding whereas the synthetic steroids STS557, RU486, ZK98.299 and ZK98.734 do not competitively inhibit the binding of [3H]-progesterone to spermatozoa. Progesterone binding sites are located in the acrosomal region of spermatozoa, as indicated by the use of fluorescein isothiocyanate-labelled progesterone-bovine serum albumin. The binding protein cross-reacts with the monoclonal antibodies to uterine progesterone receptor and has a molecular weight of approximately 85 kDa. This is the first study which demonstrates the presence of masked progesterone binding sites on the cell surface of human spermatozoa that are exposed after treatment with surfactants. These binding sites seem to have structural homology with the intracellular progesterone receptor but differ in terms of ligand specificity and location. The study further demonstrates that follicular fluid facilitates progesterone binding to spermatozoa.