The hypermodified DNA base β-d-glucosylhydroxy-methyluracil, also called J, is a naturally occurring DNA modification. J was initially detected by32P-postlabeling inTrypanosoma bruceiand was recently also found in several other eukaryotic parasites. To use32P-postlabeling as a method to quantitate the absolute levels of J in DNA we have tested the postlabeling efficiency of J using various synthesized standard oligonucleotides containing J. It is known that modified nucleotides, especially bulky ones, are often partially recovered by postlabeling and they are poor substrates for some of the enzymes used. We found that on average only 50% of J is recovered, which shows that the amount of J inT. bruceiDNA has been twofold underestimated. Experiments with a short oligomer and defined pyrimidine tracts showed that the incomplete recovery of J is caused at least in part by resistance of J-containing DNA to degradation by micrococcal nuclease.