6 -Alkylguanine-DNA alkyltransferase (AGT) is a DNA-repair protein that reverses the effects of alkylating agents by removing DNA adducts from the O 6position of guanine. We developed a real-time AGT assay that utilizes a fluorescent guanosine analog (3-methylisoxantopterin, 3-MI). 3-MI fluorescence is quenched in DNA and fluorescence intensity increases substantially with digestion of the oligonucleotide and release of 3-MI. The substrate is a doubled-stranded oligonucleotide with 3-overhangs on each end and a PvuII recognition site. PvuII is inhibited by O 6 -methylguanine, positioned within the restriction site. 3-MI is incorporated in the opposite strand just outside of the PvuII restriction site. AGT repairs O 6 -methylguanine; PvuII cleaves at its restriction site, yielding a blunt-ended double strand, which is then digested by exonuclease III. This releases 3-MI from the oligonucleotide, resulting in an increase in fluorescence intensity. All reaction components (100-L volume) are monitored in a single microcuvette. Rate of increase in fluorescence intensity is related to the amount of AGT in the reaction mixture. We measured AGT levels in extracts from a leukemia cell line, from leukemic lymphoblasts from patients, and from peripheral blood mononuclear cells from normal controls. This method may prove useful for mechanistic studies of AGT.