Epitope tags are widely used in cell biology and biochemistry research. The S-peptide/S-protein interaction has previously been utilized to purify polypeptides expressed in bacteria. We have now re-engineered the S-peptide/S-protein system to allow isolation of S-peptide-tagged polypeptides and their binding partners from eukaryotic cells with S-protein-agarose. In addition, two anti-S-peptide monoclonal antibodies have been generated for analysis of expression and subcellular localization of S-peptide-tagged polypeptides. These reagents make the S-peptide/S-protein system an attractive alternative to currently available epitope tagging methods.