A sensitive two-site enzyme immunoassay (EIA) system was established for human pS2 protein, a small estrogen-inducible secretory protein of unknown function originally identified in MCF-7 human breast cancer cells. Our EIA system is based on the sandwiching of antigen between anti-recombinant (r) pS2 antibody IgG coated on a polystyrene plate and biotinylated anti-rpS2 antibody IgG. The amount of pS2 protein was quantified by measurement of the bound enzyme activity of subsequently added streptavidin-linked β-d-galactosidase (β-d-galactosidase, EC 3.2.1.23). pS2 protein purified from MCF-7 culture supernatants was detectable at a concentration as low as 3 pg/ml (corresponding to 60 fg/well). This EIA system revealed that the amount of pS2-like immunoreactivity (LI) in human urine was 13.6 ng/mg creatinine (median, n = 416) and that there was no correlation between the pS2-LI concentration in urine and sex or aging. pS2-LI levels in plasma and sera of the normal subjects were 392 pg/ml (median, n = 14) and 494 pg/ml (median, n = 12), respectively. The serum level of the patients with breast cancer (528 pg/ml; median, n = 67) was not statistically different from that of normal subjects, although high levels of pS2 protein in breast cancer tissues had been reported.