By using a fluorescent exonuclease assay, we reported unusual electrophoretic mobility of 5'-indocarbo-cyanine 5 (5'-Cy5) labelled DNA fragments in denaturing polyacrylamide gels. Incubation time and enzyme concentration were two parameters involved in the formation of 5'-Cy5-labelled degradation products, while the structure of the substrate was slightly interfering. Replacement of positively charged 5'-Cy5-labelled DNA oligonucleotides (DNA oligos) by electrically neutral 5'-carboxyfluorescein (5'-FAM) labelled DNA oligos abolished the anomalous migration pattern of degradation products. MS analysis demonstrated that anomalously migrating products were in fact 5'-labelled DNA fragments ranging from 1 to 8 nucleotides. Longer 5'-Cy5-labelled DNA fragments migrated at the expected position. Altogether, these data highlighted, for the first time, the influence of the mass/charge ratio of 5'-Cy5-labelled DNA oligos on their electrophoretic mobility. Although obtained by performing 3' to 5' exonuclease assays with the family B DNA polymerase from Pyrococcus abyssi, these observations represent a major concern in DNA technology involving most DNA degrading enzymes.