In kinetoplastid flagellates such as Trypanosoma brucei, a small percentage of the thymine residues in the nuclear DNA is replaced by the modified base beta-D-glucosyl-hydroxymethyluracil (J), mostly in repetitive sequences like the telomeric GGGTTA repeats. In addition, traces of 5-hydroxymethyluracil (HOMeUra) are present. Previous work has suggested that J is synthesised in two steps via HOMedU as an intermediate, but as J synthesising enzymes have not yet been identified, the biosynthetic pathway remains unclear. To test a model in which HOMeUra functions as a precursor of J, we introduced an inducible gene for the human DNA glycosylase hSMUG1 into bloodstream form T.brucei. In higher eukaryotes SMUG1 excises HOMeUra as part of the base excision repair system. We show that expression of the gene in T.brucei leads to massive DNA damage in J-modified sequences and results in cell cycle arrest and, eventually, death. hSMUG1 also reduces the J content of the trypanosome DNA. This work supports the idea that HOMeUra is a precursor of J, freely accessible to a DNA glycosylase.