In plasma, protein S is found in its free form and as a complex with C4b-binding protein. After 125I-protein S was added to normal human plasma and applied to SDS-8% polyacrylamide gel electrophoresis, the autoradiogram of the gel showed only one single band at free protein S position. Applying this evidence, we have developed a peroxidase staining Western Blotting method to quantitate total protein S in human plasma which consists of sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by blotting to nitrocellulose membrane and a sensitive avidin-biotinylated peroxidase staining method (ABC technique). The measurement of protein S by the immunoblotting was reproducible and the coefficient of variation was 7%. As little as 1 ng of protein S could be detected. C4b-binding protein did not affect the measurement of protein S. Compared to other immunoassays, this peroxidase staining immunoblotting method has the advantage of directly estimating the apparent molecular weight of protein of interest, eliminating nonspecific stain and having high sensitivity without using radioisotope.