The primer oligo base extension reaction combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, recently introduced by our group for detection of single-point mutations and small insertions/deletions, has been applied to the reliable quantification of nucleotide repeat units in microsatellites. The AluVpA DNA marker within intron 5 of the interferon-alpha receptor gene was chosen as the model system. By varying the dNTP/ddNTP mixtures used, the assay could also be directed to detect the location of second-site mutations within the repeats, resulting in identification of alleles not detectable by electrophoretic sizing methods and thus an increase of the polymorphism information content for a sampling of 28 unrelated individuals. The method results in highly informative mass signals and has the potential to increase the polymorphism information content for systems containing second-site mutations; thus it is a very attractive alternative technique in statistics-based gene mapping, cancer diagnostics, and forensic applications.