Different sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) systems designed for the separation of peptides were compared for their usefulness in separating synthetic beta-amyloid peptides betaA4 (1-40) and betaA4 (1-42). Clear resolution was achieved by addition of 8 M urea to the separation gel and use of a multiphasic buffer system employing bicine and sulfate as trailing and leading ions, respectively (bicine/Tris/urea gels). Under these conditions, the longer peptide migrated faster than the one ending at amino acid 40. The usefulness of this SDS-PAGE system for the analysis of betaA4-related peptides generated during cellular metabolism was demonstrated by immunoprecipitation and electrophoretic separation of radiolabeled peptides secreted by cells transfected with amyloid precursor protein cDNAs.