High molecular weight proteins in Rattus norvegicus that are immunoreactive with an antiprotamine 2 specific antibody but not with an anti‐protamine 1 specific antibody are described. These proteins were detected by coupling high‐performance liquid chromatography (HPLC) with an enzyme‐linked immunosorbent assay (ELISA). Briefly, following HPLC separation of rat sperm nuclear proteins, the HPLC fractions were probed with the antibodies. We estimate that the antibody probes are 100–1000 times more sensitive than UV absorbance measurements. Immunoblot analysis following acid‐urea electrophoretic separation of rat sperm nuclear proteins, and of the HPLC fractions, also detected putatie protamine 2 precursor proteins. The proteins reactive with the anti‐protamine 2 antibody are most likely not mature protamine 2, since they were detected in a region of the chromatogram where we would not expect protamine 2 to migrate based on the chromatographic locations of human and mouse protamine 2. Likewise, the immunoblotting experiments demonstrated that the anti‐protamine 2 antibody recognized proteins with slower electrophoretic mobilities than would be expected for a mature protamine 2. An anti‐protamine 1 monoclonal antibody, Hup1 N, that binds rat protamine 1 is also described. Hup 1 N allowed for identification of the HPlc fractions that contained rat protamine 1. Finally, we demonstrated that Hup 1 N binds protamine 1 from a large number of species, suggesting a conserved epitope for Hup 1 N. © 1992 Wiley‐Liss, Inc.