S1 proteins (A, B, C and D) are a group of nuclear proteins, isolated by lowering pH to 4.9 of the reaction supernatant of hepatocyte nuclei that had been mildly digested with DNase I. Protein B, apparently ubiquitous in vertebrate cells, was prepared from rat liver and used to immunize a rabbit. The raised antiserum specifically reacted with S1 proteins; it reacted not only with protein B, but also with C and D. Immunoblotting demonstrated that these proteins occurred exclusively in the nucleus, being absent in the cytosol, microsome and mitochondrial fractions. Indirect immunofluorescence of liver tissue sections confirmed their nuclear localization, and further showed that the antibody selectively stained extranucleolar regions of the cell nucleus. These findings suggest that the anti-S1 antibody is specific to S1 proteins and may be useful for their structural and functional studies.