Minichromosome maintenance proteins (MCM) are required for initiation and elongation of chromosomal DNA, ensuring that DNA replication takes place only once. Although MCMs are considered of utmost importance in tumor biology and as potential marker proteins, they were not unambiguously identified at the protein level and we therefore aimed to characterize MCM 4 in a medulloblastoma cell line and provide a protein chemical analytical tool. In addition, we searched for this protein in twelve tumor cell lines and a series of non-tumor cells. The DAOY medulloblastoma cell line was cultivated, lysed, proteins extracted and run on two-dimensional gel electrophoresis with subsequent in-gel digestion and mass spectrometrical (MS/MS) analysis of protein spots. One spot at pI 6.2 with an observed molecular weight of 98 kDa was identified as minichromosome maintenance protein 4 by peptide fingerprinting. Sequence coverage of 35% along with 25 matched peptides and MS/MS analyses of three matching peptides warranted unambiguous identification. The use of mass spectrometrical identification unequivocally allowed determination of MCM 4 expression in a medulloblastoma cell line exclusively. Given the biological and probable clinical importance of this molecule as a tentative marker protein, a fair analytical tool, independent of antibody availability and specificity is mandatory and determinations at the transcriptional level cannot be extrapolated to protein levels per se, as there is a long and unpredictable way from mRNA to protein.