BALB/c mice were immunized with S-100 protein, which was isolated from bovine brain. The first fusion resulted in the cloning of three stable hybridoma lines (S1-61-64, S1-61-65, S1-87-4) that produced monoclonal antibodies against S-100 protein. The hybridoma lines obtained from a second fusion (S2-20, S2-95) were not stable and antibody production ceased early during cloning. Immunoblotting results showed that all antibodies reacted with antigenic determinants shared by both the alpha- and beta-subunit of S-100 protein. These antigenic sites appeared to differ from the calcium-binding site since immunoblotting against other calcium-binding proteins sharing this site (calmodulin, carp parvalbumin, oncomodulin) was negative. Despite the fact that the immunoblotting reactions of the antibodies obtained from both fusions were indistinguishable, different immunohistologic labeling patterns could be observed. These antibodies have proven to be excellent reagents for the immunocytochemical detection of S-100 in normal and pathologic human tissue.